Insecticidal proteins

ABSTRACT

Compositions and methods for controlling plant pests are disclosed. In particular, novel insecticidal proteins having toxicity Coleopteran and/or Lepidopteran insect pests are provided. Nucleic acid molecules encoding the novel insecticidal proteins are also provided. Methods of making the insecticidal proteins and methods of using the insecticidal proteins and nucleic acids encoding the insecticidal proteins of the invention, for example in transgenic plants to confer protection from insect damage, are also disclosed.

SEQUENCE LISTING

A Sequence Listing in ASCII text format, submitted under 37 C.F.R. §1.821, entitled “81094_ST25.txt”, 86 kilobytes in size, generated onJun. 29, 2017 and filed via EFS-Web is provided in lieu of a paper copy.This Sequence Listing is hereby incorporated by reference into thespecification for its disclosures.

FIELD OF THE INVENTION

The present invention relates to the fields of protein engineering,plant molecular biology and pest control. More particularly theinvention relates to a novel protein and its variants havinginsecticidal activity, nucleic acids whose expression results in theinsecticidal proteins, and methods of making and methods of using theinsecticidal proteins and corresponding nucleic acids to controlinsects.

BACKGROUND

Insect pests are a major cause of crop losses. In the US alone, billionsof dollars are lost every year due to infestation by various genera ofinsects. In addition to losses in field crops, insect pests are also aburden to vegetable and fruit growers, to producers of ornamentalflowers, and they are a nuisance to gardeners and homeowners.

Species of corn rootworm are considered to be the most destructive cornpests. In the United States, the three important species are Diabroticavirgifera virgifera, the western corn rootworm, D. longicornis barberi,the northern corn rootworm and D. undecimpunctata howardi, the southerncorn rootworm. Only western and northern corn rootworms are consideredprimary pests of corn in the US Corn Belt. Additionally, an importantcorn rootworm pest in the Southern US is the Mexican corn rootworm,Diabrotica virgifera zeae. Corn rootworm larvae cause the mostsubstantial plant damage by feeding almost exclusively on corn roots.This injury has been shown to increase plant lodging, to reduce grainyield and vegetative yield as well as alter the nutrient content of thegrain. Larval feeding also causes indirect effects on corn by openingavenues through the roots for bacterial and fungal infections which leadto root and stalk rot diseases. Adult corn rootworms are active incornfields in late summer where they feed on ears, silks and pollen,thus interfering with normal pollination.

Corn rootworms are mainly controlled by intensive applications ofchemical pesticides, which are active through inhibition of insectgrowth, prevention of insect feeding or reproduction, or cause death.Good corn rootworm control can thus be reached, but these chemicals cansometimes also affect other, beneficial organisms. Another problemresulting from the wide use of chemical pesticides is the appearance ofresistant insect varieties. Yet another problem is due to the fact thatcorn rootworm larvae feed underground thus making it difficult to applyrescue treatments of insecticides. Therefore, most insecticideapplications are made prophylactically at the time of planting. Thispractice results in a large environmental burden. This has beenpartially alleviated by various farm management practices, but there isan increasing need for alternative pest control mechanisms.

Biological pest control agents, such as Bacillus thuringiensis (Bt)strains expressing pesticidal toxins like δ-endotoxins(delta-endotoxins; also called crystal toxins or Cry proteins), havebeen applied to crop plants with satisfactory results against insectpests. The δ-endotoxins are proteins held within a crystalline matrixthat are known to possess insecticidal activity when ingested by certaininsects. Several native Cry proteins from Bacillus thuringiensis, orengineered Cry proteins, have been expressed in transgenic crop plantsand exploited commercially to control certain lepidopteran andcoleopteran insect pests. For example, starting in 2003, transgenic cornhybrids that control corn rootworm by expressing a Cry3Bb1,Cry34Ab1/Cry35Ab1 or modified Cry3A (mCry3A) or Cry3Ab (eCry3.1Ab)protein have been available commercially in the US.

Although the usage of transgenic plants expressing Cry proteins has beenshown to be extremely effective, insect pests that now have resistanceagainst the Cry proteins expressed in certain transgenic plants areknown. Therefore, there remains a need to identity new and effectivepest control agents that provide an economic benefit to farmers and thatare environmentally acceptable. Particularly needed are proteins thatare toxic to Diabrotica species, a major pest of corn, that have adifferent mode of action than existing insect control products as a wayto mitigate the development of resistance. Furthermore, delivery ofinsect control agents through products that minimize the burden on theenvironment, as through transgenic plants, are desirable.

SUMMARY

In view of these needs, the present invention provides novelinsecticidal proteins, namely LachbCRW, its variants, and proteins whichare substantially identical to LachbCRW and its variants. The proteinsof the invention have toxicity to corn rootworm (Diabrotica spp). Theinvention is further drawn to nucleic acid molecules that encodeLachbCRW or its variants, their complements, or which are substantiallyidentical to LachbCRW and its variants.

Also included in the invention are vectors containing such recombinant(or complementary thereto) nucleic acids; a plant or microorganism whichincludes and enables expression of such nucleic acids; plantstransformed with such nucleic acids, for example transgenic corn plants;the progeny of such plants which contain the nucleic acids stablyincorporated and hereditable in a Mendelian manner, and/or the seeds ofsuch plants and such progeny. The invention also includes methods ofbreeding to introduce a transgene comprising a nucleic acid molecule ofthe invention into a progeny plant and into various germplasms.

The invention also includes compositions and formulations containingLachbCRW or its variants, which are capable of inhibiting the ability ofinsect pests to survive, grow and/or reproduce, or of limitinginsect-related damage or loss to crop plants, for example applyingLachbCRW or its variants as part of compositions or formulations toinsect-infested areas or plants, or to prophylactically treatinsect-susceptible areas or plants to confer protection against theinsect pests.

The invention is further drawn to a method of making LachbCRW or itsvariants and to methods of using the nucleic acids, for example inmicroorganisms to control insects or in transgenic plants to conferprotection from insect damage.

The novel proteins described herein are active against insects. Forexample, in embodiments, the proteins of the present invention can beused to control economically important insect pests, includingColeopteran insects such as western corn rootworm (WCR), northern cornrootworm (NCR), southern corn rootworm (SCR) and/or Mexican cornrootworm (D. virgifera zeae). In embodiments, proteins of the presentinvention are also insecticidal against Lepidoperan insect pests such asAgrotis ipsilon (black cutworm), Diatraea saccharalis (sugar cane borer)and/or Diatraea grandiosella (southwestern corn borer). The insecticidalproteins of the invention can be used singly or in combination withother insect control strategies to confer enhanced pest controlefficiency against the same insect pest and/or to increase the spectrumof target insects with minimal environmental impact.

Other aspects and advantages of the present invention will becomeapparent to those skilled in the art from a study of the followingdescription of the invention and non-limiting examples.

BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING

SEQ ID NO: 1 is the LachbCRW amino acid sequence.

SEQ ID NO: 2 is the LachbCRW Y164W amino acid sequence

SEQ ID NO: 3 is the LachbCRW Y164F amino acid sequence.

SEQ ID NO: 4 is the LachbCRW Y164W/I169L amino acid sequence.

SEQ ID NO: 5 is the LachbCRW Y164W/Y385W amino acid sequence.

SEQ ID NO: 6 is the LachbCRW Y164W/Y400W amino acid sequence.

SEQ ID NO: 7 is the LachbCRW Y164W/Y402W amino acid sequence.

SEQ ID NO: 8 is the LachbCRW Y164W/Y431W amino acid sequence.

SEQ ID NO: 9 is the LachbCRW Y164F/I169L amino acid sequence.

SEQ ID NO: 10 is the LachbCRW Y164F/T166S amino acid sequence.

SEQ ID NO: 11 is a fragment of LachbCRW motif amino acid sequence.

SEQ ID NO: 12 is a fragment of LachbCRW Y164W amino acid sequence.

SEQ ID NO: 13 is a fragment of LachbCRW Y164F amino acid sequence.

SEQ ID NO: 14 is a fragment of LachbCRW I169L amino acid sequence.

SEQ ID NO: 15 is a fragment of LachbCRW Y164W/I169L amino acid sequence.

SEQ ID NO: 16 is a fragment of LachbCRW Y164F/I169L amino acid sequence.

SEQ ID NO: 17 is the LachbCRW nucleotide sequence.

SEQ ID NO: 18 is the LachbCRW E. coli optimized nucleotide sequence.

SEQ ID NO: 19 is the LachbCRW Y164W E. coli optimized nucleotidesequence.

SEQ ID NO: 20 is the LachbCRW Y164F E. coli optimized nucleotidesequence.

SEQ ID NO: 21 is the LachbCRW Y164W/I169L E. coli optimized nucleotidesequence.

SEQ ID NO: 22 is the LachbCRW Y164W/Y385W E. coli optimized nucleotidesequence.

SEQ ID NO: 23 is the LachbCRW Y164W/Y400W E. coli optimized nucleotidesequence.

SEQ ID NO: 24 is the LachbCRW Y164W/Y402W E. coli optimized nucleotidesequence.

SEQ ID NO: 25 is the LachbCRW Y164W/Y431W E. coli optimized nucleotidesequence.

SEQ ID NO: 26 is the LachbCRW Y164F/I169L E. coli optimized nucleotidesequence.

SEQ ID NO: 27 is the LachbCRW Y164F/T166S E. coli optimized nucleotidesequence.

SEQ ID NO: 28 is the LachbCRW maize codon-optimized nucleotide sequence.

SEQ ID NO: 29 is the LachbCRW Y164W maize codon-optimized nucleotidesequence.

SEQ ID NO: 30 is the LachbCRW Y164F maize codon-optimized nucleotidesequence.

SEQ ID NO: 31 is the LachbCRW Y164F/I169L maize codon-optimizednucleotide sequence.

SEQ ID NO: 32 is the LachbCRW Y164W/Y385W maize codon-optimizednucleotide sequence.

SEQ ID NO: 33 is the LachbCRW Y164W/Y400W maize codon-optimizednucleotide sequence.

SEQ ID NO: 34 is the LachbCRW Y164W/Y402W maize codon-optimizednucleotide sequence.

SEQ ID NO: 35 is the LachbCRW Y164W/Y431W maize codon-optimizednucleotide sequence.

SEQ ID NO: 36 is the LachbCRW Y164W/I169L maize codon-optimizednucleotide sequence.

SEQ ID NO: 37 is the LachbCRW Y164W/T166S maize codon-optimizednucleotide sequence.

Definitions

For clarity, certain terms used in the specification are defined andpresented as follows:

“Activity” of the insecticidal proteins of the invention is meant thatthe insecticidal proteins function as orally active insect controlagents, have a toxic effect, and/or are able to disrupt or deter insectfeeding, which may or may not cause death of the insect. When aninsecticidal protein of the invention is delivered to the insect, theresult is typically death of the insect, or the insect does not feedupon the source that makes the insecticidal protein available to theinsect. “Pesticidal” is defined as a toxic biological activity capableof controlling a pest, such as an insect, nematode, fungus, bacteria, orvirus, preferably by killing or destroying them. “Insecticidal” isdefined as a toxic biological activity capable of controlling insects,preferably by killing them. A “pesticidal agent” is an agent that haspesticidal activity. An “insecticidal agent” is an agent that hasinsecticidal activity.

“Associated with/operatively linked” refer to two nucleic acids that arerelated physically or functionally. For example, a promoter orregulatory DNA sequence is said to be “associated with” a DNA sequencethat codes for RNA or a protein if the two sequences are operativelylinked, or situated such that the regulatory DNA sequence will affectthe expression level of the coding or structural DNA sequence.

A “coding sequence” is a nucleic acid sequence that is transcribed intoRNA such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA.Preferably the RNA is then translated in an organism to produce aprotein.

To “control” insects means to inhibit, through a toxic effect, theability of insect pests to survive, grow, feed, and/or reproduce, or tolimit insect-related damage or loss in crop plants. To “control” insectsmay or may not mean killing the insects, although it preferably meanskilling the insects.

To “deliver” an insecticidal protein means that the insecticidal proteincomes in contact with an insect, resulting in a toxic effect and controlof the insect. The insecticidal protein may be delivered in manyrecognized ways, e.g., through a transgenic plant expressing theinsecticidal protein, formulated protein composition(s), sprayableprotein composition(s), a bait matrix, or any other art-recognized toxindelivery system.

“Effective insect-controlling amount” means that concentration of aninsecticidal protein that inhibits, through a toxic effect, the abilityof insects to survive, grow, feed and/or reproduce, or to limitinsect-related damage or loss in crop plants. “Effectiveinsect-controlling amount” may or may not mean killing the insects,although it preferably means killing the insects.

“Expression cassette” as used herein means a nucleic acid sequencecapable of directing expression of a particular nucleotide sequence inan appropriate host cell, comprising a promoter operably linked to thenucleotide sequence of interest which is operably linked to terminationsignals. It also typically comprises sequences required for propertranslation of the nucleotide sequence. The expression cassettecomprising the nucleotide sequence of interest may have at least one ofits components heterologous with respect to at least one of its othercomponents. The expression cassette may also be one that is naturallyoccurring but has been obtained in a recombinant form useful forheterologous expression. Typically, however, the expression cassette isheterologous with respect to the host, i.e., the particular nucleic acidsequence of the expression cassette does not occur naturally in the hostcell and must have been introduced into the host cell or an ancestor ofthe host cell by a transformation event. The expression of thenucleotide sequence in the expression cassette may be under the controlof a constitutive promoter or of an inducible promoter that initiatestranscription only when the host cell is exposed to some particularexternal stimulus. In the case of a multicellular organism, such as aplant, the promoter can also be specific to a particular tissue, ororgan, or stage of development.

An expression cassette comprising a nucleotide sequence of interest maybe chimeric, meaning that at least one of its components is heterologouswith respect to at least one of its other components. An expressioncassette may also be one that comprises a native promoter driving itsnative gene, however it has been obtained in a recombinant form usefulfor heterologous expression. Such usage of an expression cassette makesit so it is not naturally occurring in the cell into which it has beenintroduced.

An expression cassette also can optionally include a transcriptionaland/or translational termination region (i.e., termination region) thatis functional in plants. A variety of transcriptional terminators areavailable for use in expression cassettes and are responsible for thetermination of transcription beyond the heterologous nucleotide sequenceof interest and correct mRNA polyadenylation. The termination region maybe native to the transcriptional initiation region, may be native to theoperably linked nucleotide sequence of interest, may be native to theplant host, or may be derived from another source (i.e., foreign orheterologous to the promoter, the nucleotide sequence of interest, theplant host, or any combination thereof). Appropriate transcriptionalterminators include, but are not limited to, the CAMV 35S terminator,the tml terminator, the nopaline synthase terminator and/or the pea rbcsE9 terminator. These can be used in both monocotyledons anddicotyledons. In addition, a coding sequence's native transcriptionterminator can be used. Any available terminator known to function inplants can be used in the context of this invention.

The term “expression” when used with reference to a polynucleotide, suchas a gene, ORF or portion thereof, or a transgene in plants, refers tothe process of converting genetic information encoded in a gene into RNA(e.g., mRNA, rRNA, tRNA, or snRNA) through “transcription” of the gene(i.e., via the enzymatic action of an RNA polymerase), and into proteinwhere applicable (e.g. if a gene encodes a protein), through“translation” of mRNA. Gene expression can be regulated at many stagesin the process. For example, in the case of antisense or dsRNAconstructs, respectively, expression may refer to the transcription ofthe antisense RNA only or the dsRNA only. In embodiments, “expression”refers to the transcription and stable accumulation of sense (mRNA) orfunctional RNA. “Expression” may also refer to the production ofprotein.

A “gene” is a defined region that is located within a genome andcomprises a coding nucleic acid sequence and typically also comprisesother, primarily regulatory, nucleic acids responsible for the controlof the expression, that is to say the transcription and translation, ofthe coding portion. A gene may also comprise other 5′ and 3′untranslated sequences and termination sequences. Further elements thatmay be present are, for example, introns. The regulatory nucleic acidsequence of the gene may not normally be operatively linked to theassociated nucleic acid sequence as found in nature and thus would be achimeric gene.

“Gene of interest” refers to any nucleic acid molecule which, whentransferred to a plant, confers upon the plant a desired trait such asantibiotic resistance, virus resistance, insect resistance, diseaseresistance, or resistance to other pests, herbicide tolerance, abioticstress tolerance, male sterility, modified fatty acid metabolism,modified carbohydrate metabolism, improved nutritional value, improvedperformance in an industrial process or altered reproductive capability.The “gene of interest” may also be one that is transferred to plants forthe production of commercially valuable enzymes or metabolites in theplant.

A “heterologous” nucleic acid sequence or nucleic acid molecule is anucleic acid sequence or nucleic acid molecule not naturally associatedwith a host cell into which it is introduced, including non-naturallyoccurring multiple copies of a naturally occurring nucleic acidsequence. A heterologous nucleic acid sequence or nucleic acid moleculemay comprise a chimeric sequence such as a chimeric expression cassette,where the promoter and the coding region are derived from multiplesource organisms. The promoter sequence may be a constitutive promotersequence, a tissue-specific promoter sequence, a chemically-induciblepromoter sequence, a wound-inducible promoter sequence, astress-inducible promoter sequence, or a developmental stage-specificpromoter sequence.

A “homologous” nucleic acid sequence is a nucleic acid sequencenaturally associated with a host cell into which it is introduced.

“Homologous recombination” is the reciprocal exchange of nucleic acidfragments between homologous nucleic acid molecules.

“Identity” or “percent identity” refers to the degree of similaritybetween two nucleic acid or protein sequences. For sequence comparison,typically one sequence acts as a reference sequence to which testsequences are compared. When using a sequence comparison algorithm, testand reference sequences are input into a computer, subsequencecoordinates are designated if necessary, and sequence algorithm programparameters are designated. The sequence comparison algorithm thencalculates the percent sequence identity for the test sequence(s)relative to the reference sequence, based on the designated programparameters. The phrase “substantially identical,” in the context of twonucleic acids or two amino acid sequences, refers to two or moresequences or subsequences that have at least about 50% nucleotide oramino acid residue identity when compared and aligned for maximumcorrespondence as measured using one of the following sequencecomparison algorithms or by visual inspection. In certain embodiments,substantially identical sequences have at least about 60%, or at leastabout 70%, or at least about 80%, or at least about 85%, or even atleast about 90% or 95% nucleotide or amino acid residue identity. Incertain embodiments, substantial identity exists over a region of thesequences that is at least about 50 residues in length, or over a regionof at least about 100 residues, or the sequences are substantiallyidentical over at least about 150 residues. In further embodiments, thesequences are substantially identical when they are identical over theentire length of the coding regions.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch,J. Mol. Biol. 48: 443 (1970), by the search for similarity method ofPearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988), bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by visual inspection (seegenerally, Ausubel et al., infra).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215: 403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., 1990). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are then extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0) and N (penalty score for mismatching residues;always <0). For amino acid sequences, a scoring matrix is used tocalculate the cumulative score. Extension of the word hits in eachdirection are halted when the cumulative alignment score falls off bythe quantity X from its maximum achieved value, the cumulative scoregoes to zero or below due to the accumulation of one or morenegative-scoring residue alignments, or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison ofboth strands. For amino acid sequences, the BLASTP program uses asdefaults a wordlength (W) of 3, an expectation (E) of 10, and theBLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci.USA 89: 10915 (1989)).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90: 5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a test nucleicacid sequence is considered similar to a reference sequence if thesmallest sum probability in a comparison of the test nucleic acidsequence to the reference nucleic acid sequence is less than about 0.1,more preferably less than about 0.01, and most preferably less thanabout 0.001.

Another widely used and accepted computer program for performingsequence alignments is CLUSTALW v1.6 (Thompson, et al. Nuc. Acids Res.,22: 4673-4680, 1994). The number of matching bases or amino acids isdivided by the total number of bases or amino acids, and multiplied by100 to obtain a percent identity. For example, if two 580 base pairsequences had 145 matched bases, they would be 25 percent identical. Ifthe two compared sequences are of different lengths, the number ofmatches is divided by the shorter of the two lengths. For example, ifthere were 100 matched amino acids between a 200 and a 400 amino acidproteins, they are 50 percent identical with respect to the shortersequence. If the shorter sequence is less than 150 bases or 50 aminoacids in length, the number of matches are divided by 150 (for nucleicacid bases) or 50 (for amino acids), and multiplied by 100 to obtain apercent identity.

Another indication that two nucleic acids are substantially identical isthat the two molecules hybridize to each other under stringentconditions. The phrase “hybridizing specifically to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent conditions when that sequence ispresent in a complex mixture (e.g., total cellular) DNA or RNA. “Bind(s)substantially” refers to complementary hybridization between a probenucleic acid and a target nucleic acid and embraces minor mismatchesthat can be accommodated by reducing the stringency of the hybridizationmedia to achieve the desired detection of the target nucleic acidsequence.

“Stringent hybridization conditions” and “stringent hybridization washconditions” in the context of nucleic acid hybridization experimentssuch as Southern and Northern hybridizations are sequence dependent, andare different under different environmental parameters. Longer sequenceshybridize specifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen (1993) LaboratoryTechniques in Biochemistry and Molecular Biology-Hybridization withNucleic Acid Probes part I chapter 2 “Overview of principles ofhybridization and the strategy of nucleic acid probe assays” Elsevier,New York. Generally, highly stringent hybridization and wash conditionsare selected to be about 5° C. lower than the thermal melting point(T_(m)) for the specific sequence at a defined ionic strength and pH.Typically, under “stringent conditions” a probe will hybridize to itstarget subsequence, but to no other sequences.

The T_(m) is the temperature (under defined ionic strength and pH) atwhich 50% of the target sequence hybridizes to a perfectly matchedprobe. Very stringent conditions are selected to be equal to the T_(m)for a particular probe. An example of stringent hybridization conditionsfor hybridization of complementary nucleic acids which have more than100 complementary residues on a filter in a Southern or northern blot is50% formamide with 1 mg of heparin at 42° C., with the hybridizationbeing carried out overnight. An example of highly stringent washconditions is 0.1 5M NaCl at 72° C. for about 15 minutes. An example ofstringent wash conditions is a 0.2×SSC wash at 65° C. for 15 minutes(see, Sambrook, infra, for a description of SSC buffer). Often, a highstringency wash is preceded by a low stringency wash to removebackground probe signal. An example medium stringency wash for a duplexof, e.g., more than 100 nucleotides, is 1×SSC at 45° C. for 15 minutes.An example low stringency wash for a duplex of, e.g., more than 100nucleotides, is 4-6×SSC at 40° C. for 15 minutes. For short probes(e.g., about 10 to 50 nucleotides), stringent conditions typicallyinvolve salt concentrations of less than about 1.0 M Na ion, typicallyabout 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to8.3, and the temperature is typically at least about 30° C. Stringentconditions can also be achieved with the addition of destabilizingagents such as formamide In general, a signal to noise ratio of 2× (orhigher) than that observed for an unrelated probe in the particularhybridization assay indicates detection of a specific hybridization.Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the proteins that theyencode are substantially identical. This occurs, e.g., when a copy of anucleic acid is created using the maximum codon degeneracy permitted bythe genetic code.

The following are examples of sets of hybridization/wash conditions thatmay be used to clone homologous nucleotide sequences that aresubstantially identical to reference nucleotide sequences of the presentinvention: a reference nucleotide sequence preferably hybridizes to thereference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 MNaPO₄, 1 mM EDTA at 50° C. with washing in 2×SSC, 0.1% SDS at 50° C.,more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mMEDTA at 50° C. with washing in 1×SSC, 0.1% SDS at 50° C., more desirablystill in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50°C. with washing in 0.5×SSC, 0.1% SDS at 50° C., preferably in 7% sodiumdodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in0.1×SSC, 0.1% SDS at 50° C., more preferably in 7% sodium dodecylsulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 0.1×SSC,0.1% SDS at 65° C.

A further indication that two nucleic acids or proteins aresubstantially identical is that the protein encoded by the first nucleicacid is immunologically cross reactive with, or specifically binds to,the protein encoded by the second nucleic acid. Thus, a protein istypically substantially identical to a second protein, for example,where the two proteins differ only by conservative substitutions.

A nucleic acid sequence is “isocoding with” a reference nucleic acidsequence when the nucleic acid sequence encodes a polypeptide having thesame amino acid sequence as the polypeptide encoded by the referencenucleic acid sequence.

An “isolated” nucleic acid molecule or an isolated toxin is a nucleicacid molecule or toxin that, by the hand of man, exists apart from itsnative environment and is therefore not a product of nature. An isolatednucleic acid molecule or toxin may exist in a purified form or may existin a non-native environment such as, for example without limitation, arecombinant microbial cell, plant cell, plant tissue, or plant.

A “nucleic acid molecule” or “nucleic acid sequence” is a segment ofsingle- or double-stranded DNA or RNA that can be isolated from anysource. In the context of the present invention, the nucleic acidmolecule is typically a segment of DNA. In some embodiments, the nucleicacid molecules of the invention are isolated nucleic acid molecules.

The terms “protein,” “peptide” and “polypeptide” are usedinterchangeably herein.

As used herein, “codon optimized” sequence means the nucleotide sequenceof a recombinant, transgenic, or synthetic polynucleotide wherein thecodons are chosen to reflect the particular codon bias that a host cellmay have. This is done in such a way so as to preserve the amino acidsequence of the polypeptide encoded by the codon optimizedpolynucleotide. In certain embodiments, the nucleotide sequence of therecombinant DNA construct includes a sequence that has been codonoptimized for the cell (e.g., an animal, plant, or fungal cell) in whichthe construct is to be expressed. For example, a construct to beexpressed in a plant cell can have all or parts of its sequence (e.g.,the first gene suppression element or the gene expression element) codonoptimized for expression in a plant. See, for example, U.S. Pat. No.6,121,014, incorporated herein by reference.

A “plant” is any plant at any stage of development, particularly a seedplant.

A “plant cell” is a structural and physiological unit of a plant,comprising a protoplast and a cell wall. The plant cell may be in theform of an isolated single cell or a cultured cell, or as a part of ahigher organized unit such as, for example, plant tissue, a plant organ,or a whole plant.

“Plant cell culture” means cultures of plant units such as, for example,protoplasts, cell culture cells, cells in plant tissues, pollen, pollentubes, ovules, embryo sacs, zygotes and embryos at various stages ofdevelopment.

“Plant material” refers to leaves, stems, roots, flowers or flowerparts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell ortissue cultures, or any other part or product of a plant.

A “plant organ” is a distinct and visibly structured and differentiatedpart of a plant such as a root, stem, leaf, flower bud, or embryo.

“Plant tissue” as used herein means a group of plant cells organizedinto a structural and functional unit. Any tissue of a plant in plantaor in culture is included. This term includes, but is not limited to,whole plants, plant organs, plant seeds, tissue culture and any groupsof plant cells organized into structural and/or functional units. Theuse of this term in conjunction with, or in the absence of, any specifictype of plant tissue as listed above or otherwise embraced by thisdefinition is not intended to be exclusive of any other type of planttissue.

A “promoter” is an untranslated DNA sequence upstream of the codingregion that contains the binding site for RNA polymerase and initiatestranscription of the DNA. The promoter region may also include otherelements that act as regulators of gene expression.

“Regulatory elements” refer to sequences involved in controlling theexpression of a nucleotide sequence. Regulatory elements comprise apromoter operably linked to the nucleotide sequence of interest andtermination signals. They also typically encompass sequences requiredfor proper translation of the nucleotide sequence.

“Transformation” is a process for introducing heterologous nucleic acidinto a host cell or organism. In particular embodiments?,“transformation” means the stable integration of a DNA molecule into thegenome (nuclear or plastid) of an organism of interest.

“Transformed/transgenic/recombinant” refer to a host organism such as abacterium or a plant into which a heterologous nucleic acid molecule hasbeen introduced. The nucleic acid molecule can be stably integrated intothe genome of the host or the nucleic acid molecule can also be presentas an extrachromosomal molecule. Such an extrachromosomal molecule canbe auto-replicating. Transformed cells, tissues, or plants areunderstood to encompass not only the end product of a transformationprocess, but also transgenic progeny thereof. A “non-transformed”,“non-transgenic”, or “non-recombinant” host refers to a wild-typeorganism, e.g., a bacterium or plant, which does not contain theheterologous nucleic acid molecule.

Nucleotides are indicated by their bases by the following standardabbreviations:

adenine (A), cytosine (C), thymine (T), and guanine (G) Amino acids arelikewise indicated by the following standard abbreviations: alanine(Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp;D), cysteine (Cys; C), glutamine (Gln; Q), glutamic acid (Glu; E),glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu;L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F),proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp;W), tyrosine (Tyr; Y), and valine (Val; V).

DETAILED DESCRIPTION

This invention relates to novel insecticidal proteins which haveactivity against coleopterans, for example, Diabrotica virgiferavirgifera (western corn rootworm; WCR), Diabrotica barberi (northerncorn rootworm; NCR), and/or Diabrotica undecimpunctata howardi (southerncorn rootworm; SCR) and/or other Diabrotica species including Diabroticavirgifera zeae (Mexican corn rootworm), and/or Colorado Potato Beetle.In embodiments, a novel insecticidal protein of the invention may haveactivity against Lepidopteran species, including without limitationAgrotis ipsilon (black cutworm), Diatraea saccharalis (sugar cane borer)and/or Diatraea grandiosella (southwestern corn borer). The presentinvention also relates to nucleic acids whose expression results ininsecticidal proteins of the invention, and to the making and using ofthe insecticidal proteins to control insect pests. In embodiments, theexpression of the nucleic acids results in insecticidal proteins thatcan be used to control coleopteran insects such as western, northernand/or southern corn rootworm, particularly when expressed in atransgenic plant such as a transgenic corn plant.

The present invention further encompasses a nucleic acid moleculecomprising a nucleotide sequence that encodes an insecticidal protein ofthe invention. The nucleotide sequence may be optimized for expressionin bacteria, such as Escherichia coli, or for expression in a plant,such as Zea mays. A nucleotide sequence optimized for expression in aheterologous organism, such as a species of bacteria different fromwhere it originated or a plant, is not naturally occurring. In oneaspect of this embodiment, the nucleic acid molecule comprises anucleotide sequence selected from the group consisting of SEQ ID NO:18-37. Specifically exemplified teachings of methods to make nucleicacid molecules that encode the insecticidal proteins of the inventioncan be found in the examples of the present application. Those skilledin the art will recognize that modifications can be made to theexemplified methods to make the insecticidal proteins encompassed by thepresent invention.

A skilled person would recognize that a transgene for commercial use,such as a nucleic acid molecule that comprises any of SEQ ID NO: 17-37,may have relatively minor modifications to the nucleic acid sequence tocomply with governmental regulatory standards. Such modifications wouldnot affect the function of the resulting molecule, which would besubstantially identical to SEQ ID NO: 17-37. A skilled person wouldrecognize that the modified nucleic acid molecule would be essentiallythe same as the starting molecule, and is encompassed by the presentinvention.

The present invention also encompasses a nucleic acid molecule thatcomprises (a) a nucleotide sequence of any one of SEQ ID NOs: 17-37; (b)a nucleotide sequence that is at least 80%, at least 85%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or is 100%identical to the nucleotide sequence of any one of SEQ ID NOs: 18-37;(c) a nucleotide sequence that encodes a polypeptide, wherein the aminoacid sequence of the polypeptide comprises any one of SEQ ID NOs: 1-16,and has insect control activity; (d) a nucleotide sequence that encodesa polypeptide, wherein the amino acid sequence of the polypeptide is atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or is 100% identical to the amino acid sequenceof any one of SEQ ID NOs: 1-16; or (e) a nucleotide sequence that iscomplementary to the nucleotide sequence of any one of (a) to (d) above.

The present invention further encompasses an expression cassettecomprising a promoter operably linked to a heterologous nucleotidesequence that comprises: (a) a nucleotide sequence of any one of SEQ IDNOs: 17-37; (b) a nucleotide sequence that is at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or is 100% identical to the nucleotide sequence of any one of SEQID NOs: 18-37; (c) a nucleotide sequence that encodes a polypeptide,wherein the amino acid sequence of the polypeptide comprises any one ofSEQ ID NOs: 1-16, and has insect control activity; (d) a nucleotidesequence that encodes a polypeptide, wherein the amino acid sequence ofthe polypeptide is at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or is 100% identical tothe amino acid sequence of any one of SEQ ID NOs: 1-16; or (e) anucleotide sequence that is complementary to the nucleotide sequence ofany one of (a) to (d) above. The expression cassette comprises apromoter operably linked to a heterologous nucleotide sequence and isnot naturally occurring.

The present invention further comprises a polypeptide comprising anamino acid sequence of any one of SEQ ID NO: 2-10 or SEQ ID NO: 12-16.SEQ ID NOs: 2-10 or SEQ ID NOs: 12-16 contain engineered mutations ormodifications and the sequences are not naturally occurring. Proteinscomprising SEQ ID NOs: 2-10 or SEQ ID NO: 12-16, are exemplified in thepresent application as possessing insecticidal activity. Those skilledin the art will recognize that modifications can be made to theexemplified methods to make the insecticidal proteins encompassed by thepresent invention. Such modifications and substantially identicalnucleic acid or amino acid molecules are encompassed by the presentinvention.

The present invention also encompasses recombinant vectors orconstructs, which may also be referred to as vectors or constructs,comprising the expression cassettes and/or the nucleic acid molecules ofthis invention. In such vectors, the nucleic acids are preferably inexpression cassettes comprising regulatory elements for expression ofthe nucleotide molecules in a host cell capable of expressing thenucleotide molecules. Such regulatory elements usually comprise promoterand termination signals and preferably also comprise elements allowingefficient translation of polypeptides encoded by the nucleic acids ofthe present invention. Vectors comprising the nucleic acids are may becapable of replication in particular host cells, preferably asextrachromosomal molecules, and are therefore used to amplify thenucleic acids of this invention in the host cells. The present inventionalso encompasses a host cell that contains an expression cassette or anucleic acid molecule of the invention. In one embodiment, host cellsfor such vectors are microorganisms, such as bacteria, in particularBacillus thuringiensis or E. coli, or such as fungi such as yeast. Inanother embodiment, host cells for such recombinant vectors areendophytes or epiphytes. In yet another embodiment, such vectors areviral vectors and are used for replication of the nucleotide sequencesin particular host cells, e.g. insect cells or plant cells. Recombinantvectors are also used for transformation of the nucleotide molecules ofthis invention into host cells, whereby the nucleotide molecules arestably integrated into the DNA of a transgenic host. In one embodiment,the transgenic host is plant, for example a monocot plant, such as cornplant. In embodiments, the transgenic host plant is a dicot plant, suchas a soybean plant or cotton plant.

In another embodiment, at least one of the nucleic acids of theinvention is inserted into an appropriate expression cassette,comprising a promoter and termination signal. Expression of the nucleicacid may be constitutive, or an inducible promoter responding to varioustypes of stimuli to initiate transcription may be used. In anotherembodiment, the cell in which the insecticidal protein of the inventionis expressed is a microorganism, such as a virus, bacteria, or a fungus.In yet another embodiment, a virus, such as a baculovirus, contains anucleic acid of the invention in its genome and expresses large amountsof the corresponding insecticidal protein after infection of appropriateeukaryotic cells that are suitable for virus replication and expressionof the nucleic acid. The insecticidal protein thus produced is used asan insecticidal agent. Alternatively, baculoviruses engineered toinclude the nucleic acid are used to infect insects in vivo and killthem either by expression of the insecticidal toxin or by a combinationof viral infection and expression of the insecticidal toxin. In afurther embodiment, the present invention also encompasses a method forproducing a polypeptide with insecticidal activity, comprising culturingthe host cell under conditions in which the nucleic acid moleculeencoding the polypeptide is expressed.

Bacterial cells are also hosts for the expression of the nucleic acidsof the invention. In one embodiment, non-pathogenic symbiotic bacteria,which are able to live and replicate within plant tissues, so-calledendophytes, or non-pathogenic symbiotic bacteria, which are capable ofcolonizing the phyllosphere or the rhizosphere, so-called epiphytes, areused. Such bacteria include bacteria of the genera Agrobacterium,Alcaligenes, Azospirillum, Azotobacter, Bacillus, Clavibacter,Enterobacter, Erwinia, Flavobacter, Klebsiella, Pseudomonas, Rhizobium,Serratia, Streptomyces and Xanthomonas. Symbiotic fungi, such asTrichoderma and Gliocladium are also possible hosts for expression ofthe inventive nucleic acids for the same purpose.

Techniques for these genetic manipulations are specific for thedifferent available hosts and are known in the art. For example, theexpression vectors pKK223-3 and pKK223-2 can be used to expressheterologous genes in E. coli, either in transcriptional ortranslational fusion, behind the tac or trc promoter. For the expressionof operons encoding multiple ORFs, the simplest procedure is to insertthe operon into a vector such as pKK223-3 in transcriptional fusion,allowing the cognate ribosome binding site of the heterologous genes tobe used. Techniques for overexpression in gram-positive species such asBacillus are also known in the art and can be used in the context ofthis invention (Quax et al. In: Industrial Microorganisms:Basic andApplied Molecular Genetics, Eds. Baltz et al., American Society forMicrobiology, Washington (1993)). Alternate systems for overexpressionrely for example, on yeast vectors and include the use of Pichia,Saccharomyces and Kluyveromyces (Sreekrishna, In: Industrialmicroorganisms:basic and applied molecular genetics, Baltz, Hegeman, andSkatrud eds., American Society for Microbiology, Washington (1993);Dequin & Barre, Biotechnology L2:173-177 (1994); van den Berg et al.,Biotechnology 8:135-139 (1990)).

The insecticidal proteins of the present invention have insect controlactivity when tested against insect pests in bioassays. In oneembodiment, the insecticidal proteins of the invention are activeagainst coleopteran and/or lepidopteran insects. Insects in the orderLepidoptera include without limitation any insect now known or lateridentified that is classified as a lepidopteran, including those insectspecies within suborders Zeugloptera, Glossata, and Heterobathmiina, andany combination thereof. Exemplary lepidopteran insects include, but arenot limited to, Ostrinia spp. such as O. nubilalis (European cornborer); Plutella spp. such as P. xylostella (diamondback moth);Spodoptera spp. such as S. frugiperda (fall armyworm), S. ornithogalli(yellowstriped armyworm), S. praefica (western yellowstriped armyworm),S. eridania (southern armyworm) and S. exigua (beet armyworm); Agrotisspp. such as A. ipsilon (black cutworm), A. segetum (common cutworm), A.gladiaria (claybacked cutworm), and A. orthogonia (pale westerncutworm); Striacosta spp. such as S. albicosta (western bean cutworm);Helicoverpa spp. such as H. zea (corn earworm), H. punctigera (nativebudworm), S. littoralis (Egyptian cotton leafworm) and H. armigera(cotton bollworm); Heliothis spp. such as H. virescens (tobaccobudworm); Diatraea spp. such as D. grandiosella (southwestern cornborer) and D. saccharalis (sugarcane borer); Trichoplusia spp. such asT. ni (cabbage looper); Sesamia spp. such as S. nonagroides(Mediterranean corn borer); Pectinophora spp. such as P. gossypiella(pink bollworm); Cochylis spp. such as C. hospes (banded sunflowermoth); Manduca spp. such as M. sexta (tobacco hornworm) and M.quinquemaculata (tomato hornworm); Elasmopalpus spp. such as E.lignosellus (lesser cornstalk borer); Pseudoplusia spp. such as P.includens (soybean looper); Anticarsia spp. such as A. gemmatalis(velvetbean caterpillar); Plathypena spp. such as P. scabra (greencloverworm); Pieris spp. such as P. brassicae (cabbage butterfly),Papaipema spp. such as P. nebris (stalk borer); Pseudaletia spp. such asP. unipuncta (common armyworm); Peridroma spp. such as P. saucia(variegated cutworm); Keiferia spp. such as K. lycopersicella (tomatopinworm); Artogeia spp. such as A. rapae (imported cabbageworm);Phthorimaea spp. such as P. operculella (potato tuberworm); Crymodesspp. such as C. devastator (glassy cutworm); Feltia spp. such as F.ducens (dingy cutworm); and any combination of the foregoing. In oneaspect of this embodiment, the insecticidal proteins of the inventionare active against black cutworm, sugar cane borer, and/or southwesterncorn borer.

Insects in the order Coleoptera include but are not limited to anycoleopteran insect now known or later identified including those insuborders Archostemata, Myxophaga, Adephaga and Polyphaga, and anycombination thereof.

In one aspect of this embodiment, the insecticidal proteins of theinvention are active against Diabrotica spp. Diabrotica is a genus ofbeetles of the order Coleoptera commonly referred to as “corn rootworms”or “cucumber beetles.” Exemplary Diabrotica species include withoutlimitation Diabrotica barberi (northern corn rootworm), D. virgiferavirgifera (western corn rootworm), D. undecimpunctata howardii (southerncorn rootworm), D. balteata (banded cucumber beetle), D. undecimpunctataundecimpunctata (western spotted cucumber beetle), D. significata(3-spotted leaf beetle), D. speciosa (chrysanthemum beetle), D.virgifera zeae (Mexican corn rootworm), D. beniensis, D. cristata, D.curviplustalata, D. dissimilis, D. elegantula, D. emorsitans, D.graminea, D. hispanloe, D. lemniscata, D. linsleyi, D. milleri, D.nummularis, D. occlusal, D. porrecea, D. scutellata, D. tibialis, D.trifasciata and D. viridula; and any combination thereof.

Other nonlimiting examples of Coleopteran insect pests according to thepresent invention include Leptinotarsa spp. such as L. decemlineata(Colorado potato beetle); Chrysomela spp. such as C. scripta (cottonwoodleaf beetle); Hypothenemus spp. such as H. hampei (coffee berry borer);Sitophilus spp. such as S. zeamais (maize weevil); Epitrix spp. such asE. hirtipennis (tobacco flea beetle) and E. cucumeris (potato fleabeetle); Phyllotreta spp. such as P. cruciferae (crucifer flea beetle)and P. pusilla (western black flea beetle); Anthonomus spp. such as A.eugenii (pepper weevil); Hemicrepidus spp. such as H. memnonius(wireworms); Melanotus spp. such as M. communis (wireworm); Ceutorhychusspp. such as C. assimilis (cabbage seedpod weevil); Phyllotreta spp.such as P. cruciferae (crucifer flea beetle); Aeolus spp. such as A.mellillus (wireworm); Aeolus spp. such as A. mancus (wheat wireworm);Horistonotus spp. such as H. uhlerii (sand wireworm); Sphenophorus spp.such as S. maidis (maize billbug), S. zeae (timothy billbug), S.parvulus (bluegrass billbug), and S. callosus (southern corn billbug);Phyllophaga spp. (White grubs); Chaetocnema spp. such as C. pulicaria(corn flea beetle); Popillia spp. such as P. japonica (Japanese beetle);Epilachna spp. such as E. varivestis (Mexican bean beetle); Cerotomaspp. such as C. trifurcate (Bean leaf beetle); Epicauta spp. such as E.pestifera and E. lemniscata (Blister beetles); and any combination ofthe foregoing.

The insecticidal proteins of the invention may also be active againstHemipteran, Dipteran, Lygus spp., and/or other piercing and suckinginsects, for example of the order Orthoptera or Thysanoptera. Insects inthe order Diptera include but are not limited to any dipteran insect nowknown or later identified including but not limited to Liriomyza spp.such as L. trifolii (leafminer) and L. sativae (vegetable leafminer);Scrobipalpula spp. such as S. absoluta (tomato leafminer); Delia spp.such as D. platura (seedcorn maggot), D. brassicae (cabbage maggot) andD. radicum (cabbage root fly); Psilia spp. such as P. rosae (carrot rustfly); Tetanops spp. such as T. myopaeformis (sugarbeet root maggot); andany combination of the foregoing.

Insects in the order Orthoptera include but are not limited to anyorthopteran insect now known or later identified including but notlimited to Melanoplus spp. such as M. differentialis (Differentialgrasshopper), M. femurrubrum (Redlegged grasshopper), M. bivittatus(Twostriped grasshopper); and any combination thereof.

Insects in the order Thysanoptera include but are not limited to anythysanopteran insect now known or later identified including but notlimited to Frankliniella spp. such as F. occidentalis (western flowerthrips) and F. fusca (tobacco thrips); and Thrips spp. such as T. tabaci(onion thrips), T. palmi (melon thrips); and any combination of theforegoing.

The insecticidal proteins of the invention may also be active againstnematodes. The term “nematode” as used herein encompasses any organismthat is now known or later identified that is classified in the animalkingdom, phylum Nematoda, including without limitation nematodes withinclass Adenophorea (including for example, orders Enoplida, Isolaimida,Mononchida, Dorylaimida, Trichocephalida, Mermithida, Muspiceida,Araeolaimida, Chromadorida, Desmoscolecida, Desmodorida andMonhysterida) and/or class Secernentea (including, for example, ordersRhabdita, Strongylida, Ascaridida, Spirurida, Camallanida,Diplogasterida, Tylenchida and Aphelenchida).

Nematodes include but are not limited to parasitic nematodes such asroot-knot nematodes, cyst nematodes and/or lesion nematodes. Exemplarygenera of nematodes according to the present invention include but arenot limited to, Meloidogyne (root-knot nematodes), Heterodera (cystnematodes), Globodera (cyst nematodes), Radopholus (burrowingnematodes), Rotylenchulus (reniform nematodes), Pratylenchus (lesionnematodes), Aphelenchoides (foliar nematodes), Helicotylenchus (spiralnematodes), Hoplolaimus (lance nematodes), Paratrichodorus (stubby-rootnematodes), Longidorus, Nacobbus (false root-knot nematodes),Subanguina, Belonlaimus (sting nematodes), Criconemella, Criconemoides(ring nematodes), Ditylenchus, Dolichodorus, Hemicriconemoides,Hemicycliophora, Hirschmaniella, Hypsoperine, Macroposthonia, Melinius,Punctodera, Quinisulcius, Scutellonema, Xiphinema (dagger nematodes),Tylenchorhynchus (stunt nematodes), Tylenchulus, Bursaphelenchus (roundworms), and any combination thereof.

Exemplary plant parasitic nematodes according to the present inventioninclude, but are not limited to, Belonolaimus gracilis, Belonolaimuslongicaudatus, Bursaphelenchus xylophilus (pine wood nematode),Criconemoides ornata, Ditylenchus destructor (potato rot nematode),Ditylenchus dipsaci (stem and bulb nematode), Globodera pallida (potatocyst nematode), Globodera rostochiensis (golden nematode), Heteroderaglycines (soybean cyst nematode), Heterodera schachtii (sugar beet cystnematode); Heterodera zeae (corn cyst nematode), Heterodera avenae(cereal cyst nematode), Heterodera carotae, Heterodera trifolii,Hoplolaimus columbus, Hoplolaimus galeatus, Hoplolaimus magnistylus,Longidorus breviannulatus, Meloidogyne arenaria, Meloidogyne chitwoodi,Meloidogyne hapla, Meloidogyne incognita, Meloidogyne javanica,Mesocriconema xenoplax, Nacobbus aberrans, Naccobus dorsalis,Paratrichodorus christiei, Paratrichodorus minor, Pratylenchusbrachyurus, Pratylenchus crenatus, Pratylenchus hexincisus, Pratylenchusneglectus, Pratylenchus penetrans, Pratylenchus projectus, Pratylenchusscribneri, Pratylenchus tenuicaudatus, Pratylenchus thornei,Pratylenchus zeae, Punctodera chaccoensis, Quinisulcius acutus,Radopholus similis, Rotylenchulus reniformis, Tylenchorhynchus dubius,Tylenchulus semipenetrans (citrus nematode), Siphinema americanum, X.Mediterraneum, and any combination of the foregoing.

In another embodiment, the invention encompasses a method of producing ainsecticidal protein that is active against insects, comprising: (a)obtaining a host cell comprising a gene, which itself comprises anexpression cassette and/or a nucleic acid molecule of the invention; and(b) growing the transgenic host cell in such a manner to express aninsecticidal protein that is active against insects.

In yet a further embodiment, the invention encompasses a method ofcontrolling insects, comprising delivering to the insects an effectiveinsect-controlling amount of an insecticidal protein of the invention.

In one embodiment, at least one of the insecticidal proteins of theinvention is expressed in a higher organism such as a plant. In thiscase, transgenic plants expressing effective insect-controlling amountsof the insecticidal protein protect themselves from insect pests. Whenthe insect starts feeding on such a transgenic plant, it also ingeststhe expressed insecticidal protein. This will deter the insect fromfurther biting into the plant tissue and/or may even harm or kill theinsect. A nucleic acid of the present invention is inserted into anexpression cassette, which may then be stably integrated in the genomeof the plant. In another embodiment, the nucleic acid is included in anon-pathogenic self-replicating virus. Plants transformed in accordancewith the present invention may be monocotyledonous or dicotyledonous andinclude, but are not limited to, corn, wheat, oat, turfgrass, pasturegrass, flax, barley, rye, sweet potato, bean, pea, chicory, lettuce,cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus,onion, garlic, pepper, celery, squash, pumpkin, hemp, zucchini, apple,pear, quince, melon, plum, cherry, peach, nectarine, apricot,strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya,mango, banana, soybean, tomato, sorghum, sugarcane, sugar beet,sunflower, rapeseed, clover, tobacco, carrot, cotton, alfalfa, rice,potato, eggplant, cucumber, Arabidopsis, and woody plants such asconiferous and deciduous trees.

In another embodiment, the invention encompasses a method of producing aplant or plant part having enhanced insect resistance as compared to acontrol plant or plant part, comprising: (a) introducing a nucleic acidmolecule comprising an expression cassette of the invention; and (b)growing the plant part into a plant that expresses the heterologousnucleic acid molecule of the expression cassette and that has enhancedinsect resistance as compared to a control plant or plant part that hasnot been transformed with a nucleic acid molecule comprising theexpression cassette. In a preferred embodiment, the expression cassettemay encode a polypeptide comprising an amino acid sequence that is atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or is 100% identical or similar to any one ofSEQ ID NO: 1-16. In a preferred embodiment, the expression cassette mayencode a polypeptide comprising an amino acid sequence that is at least80% identical to SEQ ID NO: 9. “Enhanced” insect resistance may bemeasured as an increase insecticidal activity Enhanced insect resistancemay be greater than 0%, at least 1%, at least 2%, at least 3%, at least4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%,at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 100%, at least 125%, at least 150%, atleast 200%, at least 300%, at least 400%, at least 500%, at least 600%,at least 700%, at least 800%, at least 900%, or at least 1000% greaterinsecticidal activity compared to a control plant. A plant or plant parthaving enhance insect resistance as compared to a control plant or plantpart may be produced by methods of plant transformation, plant tissueculture, or breeding. The plant or plant part may be produced by methodsof sexual or asexual propagation. Any suitable control plant or plantpart can be used, for example a plant of the same or similar geneticbackground grown in the same environment. In embodiments, the controlplant or plant part is of the same genetic background and is growing inthe same environment as the described plant, but it does not comprise amolecule of the invention, while the described plant does comprise amolecule of the invention.

In another embodiment, the invention encompasses a method of enhancinginsect resistance in a plant or plant part as compared to a controlplant or plant part, comprising expressing in the plant or plant part anucleic acid molecule or an expression cassette of the invention,wherein expression of the heterologous nucleic acid of the expressioncassette results in enhanced insect resistance in a plant or plant partas compared to a control plant or plant part. In embodiments, theexpression cassette or nucleic acid molecule comprises a promoteroperably linked to a heterologous nucleic acid molecule comprising anucleotide sequence that comprises: (a) a nucleotide sequence of any oneof SEQ ID NOs: 18-37; (b) a nucleotide sequence that is at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or is 100% identical to the nucleotide sequence of any one ofSEQ ID NOs: 18-37; (c) a nucleotide sequence that encodes a polypeptide,wherein the amino acid sequence of the polypeptide comprises any one ofSEQ ID NOs: 1-16, and has insect control activity; (d) a nucleotidesequence that encodes a polypeptide, wherein the amino acid sequence ofthe polypeptide is at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or is 100% identical tothe amino acid sequence of any one of SEQ ID NOs: 1-16; or (e) anucleotide sequence that is complementary to the nucleotide sequence ofany one of (a) to (d) above. The nucleic acid molecule or expressioncassette may be introduced into the plant. In some embodiments, thenucleic acid molecule or expression cassette may be introduced into aplant part and a plant comprising the nucleic acid molecule orexpression cassette may be produced from the plant part.

In another embodiment, the invention encompasses a method of producing aplant having enhanced insect resistance as compared to a control plant,comprising detecting, in a plant part, a heterologous nucleic acidcomprising a nucleic acid molecule or an expression cassette of theinvention and producing a plant from the plant part, thereby producing aplant having enhanced insect resistance as compared to a control plant.In a further embodiment, the invention encompasses a method ofidentifying a plant or plant part having enhanced insect resistance ascompared to a control plant or plant part, comprising detecting, in theplant or plant part, a nucleic acid molecule or an expression cassetteof the invention, thereby identifying a plant or plant part havingenhanced insect resistance. In a further embodiment, the expressioncassette or a diagnostic fragment thereof is detected in anamplification product from a nucleic acid sample from the plant or plantpart. The diagnostic fragment may be a nucleic acid molecule at least 10contiguous nucleotides long which is unique to the expression cassetteof the invention.

In yet another embodiment, the invention encompasses a method ofproducing a plant having enhanced insect resistance as compared to acontrol plant or plant part, comprising crossing a first parent plantwith a second parent plant, wherein at least the first parent plantcomprises within its genome a heterologous nucleic acid that comprises anucleic acid molecule or an expression cassette of the invention andproducing a progeny generation, wherein the progeny generation comprisesat least one plant that possesses the heterologous nucleic acid withinits genome and that exhibits enhanced insect resistance as compared to acontrol plant.

In preferred embodiments, the methods of the invention confer enhancedinsect resistance in a plant or plant part against a coleopteran and/ora lepidopteran insect pest. Insect control of both lepidopteran andcoleopteran insect pests are demonstrated in the Examples. In furtherembodiments, the methods of the invention confer enhanced insectresistance in a plant or plant part against Diabrotica species,including Diabrotica virgifera virgifera, Diabrotica barberi, Diabroticaundecimpunctata howardi, Diabrotica virgifera zeae, and/or Diabroticaspeciosa, and/or related species. In further embodiments, the methods ofthe invention confer enhanced insect resistance in a plant or plant partagainst Diabrotica virgifera virgifera, Diabrotica barberi, and/orDiabrotica undecimpunctata howardi.

In preferred embodiments, the methods of the invention confer enhancedinsect resistance in a monocotyledonous plant.

The present invention further encompasses a transgenic plant comprisinga a heterologous nucleic acid molecule or an expression cassette of theinvention, which when transcribed and translated confers enhanced insectresistance. In preferred embodiments, the heterologous nucleic acidmolecule comprises a sequence at least 80%, at least 85%, at least 90%,at least 91% at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% at least 99%, or 100% identical toany one of SEQ ID NOs: 17-37. In a further embodiment, the transgenicplant comprises a heterologous nucleic acid molecule comprising asequence at least 80%, at least 85%, at least 90%, at least 91% at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98% at least 99%, or 100% identical to SEQ ID NO: 37. Inembodiments, the transgenic plant is a dicotyledonous plant. Inpreferred embodiments, the transgenic plant is a monocotyledonous plant.In further embodiments, the transgenic plant is alfalfa, aneth, apple,apricot, artichoke, arugula, asparagus, avocado, banana, beans, beet,blackberry, blueberry, broccoli, brussel sprouts, cabbage, canola,cantaloupe, carrot, cassava, cauliflower, celery, cherry, cilantro,citrus, clementine, coffee, corn, cotton, cucumber, Douglas fir,eggplant, endive, escarole, eucalyptus, fennel, figs, gourd, grape,grapefruit, honey dew, jicama, kiwifruit, lettuce, leeks, lemon, lime,Loblolly pine, mango, melon, mushroom, nut, okra, onion, orange, anornamental plant, papaya, parsley, pea, peach, peanut, pear, pepper,persimmon, pine, pineapple, plantain, plum, pomegranate, poplar, potato,pumpkin, quince, radiata pine, radicchio, radish, raspberry, rice, rye,sorghum, Southern pine, soybean, spinach, squash, strawberry, sugarbeet,sunflower, sweet potato, sweetgum, tangerine, tea, tobacco, tomato,turf, a vine, watermelon, yams, or zucchini. In preferred embodiments,the transgenic plant is millet, switchgrass, maize, sorghum, wheat, oat,turf grass, pasture grass, flax, rice, sugarcane, oilseed rape, orbarley.

In yet another embodiment, a transgenic plant of the invention comprisesa heterologous nucleic acid molecule comprising a promoter sequence. Inyet another embodiment, a transgenic plant of the invention may comprisea heterologous nucleic acid molecule which encodes for at least oneadditional desired trait. The additional trait may be encoded on thesame heterologous nucleic acid molecule as a molecule of the invention,or it may be encoded on a second heterologous nucleic acid molecule. Theadditional desired trait may confer insect resistance to a second insectpest, insect resistance to the same insect pest, abiotic stresstolerance, male sterility, herbicide resistance, bacterial diseaseresistance, fungal disease resistance, viral disease resistance,nematode resistance, modified fatty acid metabolism, modifiedcarbohydrate metabolism, improved nutritional value, improvedperformance in an industrial process or altered reproductive capability.The additional desired trait may also induce production within the plantof a commercially valuable enzyme or metabolite.

In embodiments, the desired added trait is a second pesticidal agent.The second pesticidal agent may be active on any plant pest, includinginsects, nematodes, fungi, viruses or bacteria. Examples of insect plantpests include and are not limited to Nilaparvata spp. (e.g. N. lugens(brown planthopper)); Laodelphax spp. (e.g. L. striatellus (small brownplanthopper)); Nephotettix spp. (e.g. N. virescens or N. cincticeps(green leafhopper), or N. nigropictus (rice leafhopper)); Sogatella spp.(e.g. S. furcifera (white-backed planthopper)); Blissus spp. (e.g. B.leucopterus leucopterus (chinch bug)); Scotinophora spp. (e.g. S.vermidulate (rice blackbug)); Acrosternum spp. (e.g. A. hilare (greenstink bug)); Parnara spp. (e.g. P. guttata (rice skipper)); Chilo spp.(e.g. C. suppressalis (rice striped stem borer), C. auricilius(gold-fringed stem borer), or C. polychrysus (dark-headed stem borer));Chilotraea spp. (e.g. C. polychrysa (rice stalk borer)); Sesamia spp.(e.g. S. inferens (pink rice borer)); Tryporyza spp. (e.g. T. innotata(white rice borer), or T. incertulas (yellow rice borer));Cnaphalocrocis spp. (e.g. C. medinalis (rice leafroller)); Agromyza spp.(e.g. A. oryzae (leafminer), or A. parvicornis (corn blot leafminer));Diatraea spp. (e.g. D. saccharalis (sugarcane borer), or D. grandiosella(southwestern corn borer)); Narnaga spp. (e.g. N. aenescens (green ricecaterpillar)); Xanthodes spp. (e.g. X. transversa (green caterpillar));Spodoptera spp. (e.g. S. frugiperda (fall armyworm), S. exigua (beetarmyworm), S. littoralis (climbing cutworm) or S. praefica (westernyellowstriped armyworm)); Mythimna spp. (e.g. Mythmna (Pseudaletia)seperata (armyworm)); Helicoverpa spp. (e.g. H. zea (corn earworm));Colaspis spp. (e.g. C. brunnea (grape colaspis)); Lissorhoptrus spp.(e.g. L. oryzophilus (rice water weevil)); Echinocnemus spp. (e.g. E.squamos (rice plant weevil)); Diclodispa spp. (e.g. D. armigera (ricehispa)); Oulema spp. (e.g. O. oryzae (leaf beetle); Sitophilus spp.(e.g. S. oryzae (rice weevil)); Pachydiplosis spp. (e.g. P. oryzae (ricegall midge)); Hydrellia spp. (e.g. H. griseola (small rice leafminer),or H. sasakii (rice stem maggot)); Chlorops spp. (e.g. C. oryzae (stemmaggot)); Diabrotica spp. (e.g. D. virgifera virgifera (western cornrootworm), D. barberi (northern corn rootworm), D. undecimpunctatahowardi (southern corn rootworm), D. virgifera zeae (Mexican cornrootworm); D. balteata (banded cucumber beetle)); Ostrinia spp. (e.g. O.nubilalis (European corn borer)); Agrotis spp. (e.g. A. ipsilon (blackcutworm)); Elasmopalpus spp. (e.g. E. lignosellus (lesser cornstalkborer)); Melanotus spp. (wireworms); Cyclocephala spp. (e.g. C. borealis(northern masked chafer), or C. immaculata (southern masked chafer));Popillia spp. (e.g. P. japonica (Japanese beetle)); Chaetocnema spp.(e.g. C. pulicaria (corn flea beetle)); Sphenophorus spp. (e.g. S.maidis (maize billbug)); Rhopalosiphum spp. (e.g. R. maidis (corn leafaphid)); Anuraphis spp. (e.g. A. maidiradicis (corn root aphid));Melanoplus spp. (e.g. M. femurrubrum (redlegged grasshopper) M.differentialis (differential grasshopper) or M. sanguinipes (migratorygrasshopper)); Hylemya spp. (e.g. H. platura (seedcorn maggot));Anaphothrips spp. (e.g. A. obscrurus (grass thrips)); Solenopsis spp.(e.g. S. milesta (thief ant)); or spp. (e.g. T. urticae (twospottedspider mite), T. cinnabarinus (carmine spider mite); Helicoverpa spp.(e.g. H. zea (cotton bollworm), or H. armigera (American bollworm));Pectinophora spp. (e.g. P. gossypiella (pink bollworm)); Earias spp.(e.g. E. vittella (spotted bollworm)); Heliothis spp. (e.g. H. virescens(tobacco budworm)); Anthonomus spp. (e.g. A. grandis (boll weevil));Pseudatomoscelis spp. (e.g. P. seriatus (cotton fleahopper));Trialeurodes spp. (e.g. T. abutiloneus (banded-winged whitefly) T.vaporariorum (greenhouse whitefly)); Bemisia spp. (e.g. B. argentifolii(silverleaf whitefly)); Aphis spp. (e.g. A. gossypii (cotton aphid));Lygus spp. (e.g. L. lineolaris (tarnished plant bug) or L. hesperus(western tarnished plant bug)); Euschistus spp. (e.g. E. conspersus(consperse stink bug)); Chlorochroa spp. (e.g. C. sayi (Say stinkbug));Nezara spp. (e.g. N. viridula (southern green stinkbug)); Thrips spp.(e.g. T. tabaci (onion thrips)); Frankliniella spp. (e.g. F. fusca(tobacco thrips), or F. occidentalis (western flower thrips));Leptinotarsa spp. (e.g. L. decemlineata (Colorado potato beetle), L.juncta (false potato beetle), or L. texana (Texan false potato beetle));Lema spp. (e.g. L. trilineata (three-lined potato beetle)); Epitrix spp.(e.g. E. cucumeris (potato flea beetle), E. hirtipennis (flea beetle),or E. tuberis (tuber flea beetle)); Epicauta spp. (e.g. E. vittata(striped blister beetle)); Phaedon spp. (e.g. P. cochleariae (mustardleaf beetle)); Epilachna spp. (e.g. E. varivetis (mexican bean beetle));Acheta spp. (e.g. A. domesticus (house cricket)); Empoasca spp. (e.g. E.fabae (potato leafhopper)); Myzus spp. (e.g. M. persicae (green peachaphid)); Paratrioza spp. (e.g. P. cockerelli (psyllid)); Conoderus spp.(e.g. C. falli (southern potato wireworm), or C. vespertinus (tobaccowireworm)); Phthorimaea spp. (e.g. P. operculella (potato tuberworm));Macrosiphum spp. (e.g. M. euphorbiae (potato aphid)); Thyanta spp. (e.g.T. pallidovirens (redshouldered stinkbug)); Phthorimaea spp. (e.g. P.operculella (potato tuberworm)); Helicoverpa spp. (e.g. H. zea (tomatofruitworm); Keiferia spp. (e.g. K. lycopersicella (tomato pinworm));Limonius spp. (wireworms); Manduca spp. (e.g. M. sexta (tobaccohornworm), or M. quinquemaculata (tomato hornworm)); Liriomyza spp.(e.g. L. sativae, L. trifolli or L. huidobrensis (leafminer));Drosophilla spp. (e.g. D. melanogaster, D. yakuba, D. pseudoobscura orD. simulans); Carabus spp. (e.g. C. granulatus); Chironomus spp. (e.g.C. tentanus); Ctenocephalides spp. (e.g. C. felis (cat flea)); Diaprepesspp. (e.g. D. abbreviatus (root weevil)); Ips spp. (e.g. I. pini (pineengraver)); Tribolium spp. (e.g. T. castaneum (red floor beetle));Glossina spp. (e.g. G. morsitans (tsetse fly)); Anopheles spp. (e.g. A.gambiae (malaria mosquito)); Helicoverpa spp. (e.g. H. armigera (AfricanBollworm)); Acyrthosiphon spp. (e.g. A. pisum (pea aphid)); Apis spp.(e.g. A. melifera (honey bee)); Homalodisca spp. (e.g. H. coagulate(glassy-winged sharpshooter)); Aedes spp. (e.g. Ae. aegypti (yellowfever mosquito)); Bombyx spp. (e.g. B. mori (silkworm)); Locusta spp.(e.g. L. migratoria (migratory locust)); Boophilus spp. (e.g. B.microplus (cattle tick)); Acanthoscurria spp. (e.g. A. gomesiana(red-haired chololate bird eater)); Diploptera spp. (e.g. D. punctata(pacific beetle cockroach)); Heliconius spp. (e.g. H. erato (red passionflower butterfly) or H. melpomene (postman butterfly)); Curculio spp.(e.g. C. glandium (acorn weevil)); Plutella spp. (e.g. P. xylostella(diamondback moth)); Amblyomma spp. (e.g. A. variegatum (cattle tick));Anteraea spp. (e.g. A. yamamai (silkmoth)); and Armigeres spp. (e.g. A.subalbatus).

The insecticidal proteins of the invention can be used in combinationwith other pesticidal agents (e.g. Bt Cry proteins) to increase pesttarget range. Furthermore, the use of the insecticidal proteins of theinvention in combination with an insecticidal agent which has adifferent mode of action or target a different receptor in the insectgut has particular utility for the prevention and/or management ofinsect resistance.

The second pesticidal agent may be an insecticidal protein derived fromBacillus thuringiensis. A B. thuringiensis insecticidal protein can beany of a number of insecticidal proteins including but not limited to aCry1 protein, a Cry3 protein, a Cry7 protein, a Cry8 protein, a Cry11protein, a Cry22 protein, a Cry 23 protein, a Cry 36 protein, a Cry37protein, a Cry34 protein together with a Cry35 protein, a binaryinsecticidal protein CryET33 and CryET34, a binary insecticidal proteinTIC100 and TIC101, a binary insecticidal protein PS149B1, a VIP(Vegetative Insecticidal Protein, disclosed in U.S. Pat. Nos. 5,849,870and 5,877,012, herein incorporated by reference), a TIC900 or relatedprotein, a TIC901, TIC1201, TIC407, TIC417, a modified Cry3A protein, orhybrid proteins or chimeras made from any of the preceding insecticidalproteins. In other embodiments, the B. thuringiensis insecticidalprotein is selected from the group consisting of Cry3Bb1, Cry34Ab1together with Cry35Ab1, mCry3A (U.S. Pat. No. 7,276,583, incorporated byreference herein), eCry3.1Ab (U.S. Pat. No. 8,309,516, incorporated byreference herein), and Vip3A proteins, including Vip3Aa (U.S. Pat. No.6,137,033, incorporated by reference herein).

In other embodiments, a transgenic plant of the invention may comprise asecond pesticidal agent which may be derived from sources other than B.thuringiensis. The second insecticidal agent can be an agent selectedfrom the group comprising an a amylase, a peroxidase, a cholesteroloxidase, a patatin, a protease, a protease inhibitor, a urease, analpha-amylase inhibitor, a pore-forming protein, a chitinase, a lectin,an engineered antibody or antibody fragment, a Bacillus cereusinsecticidal protein, a Xenorhabdus spp. (such as X. nematophila or X.bovienii) insecticidal protein, a Photorhabdus spp. (such as P.luminescens or P. asymobiotica) insecticidal protein, a Brevibacillusspp. (such as B. laterosporous) insecticidal protein, a Lysinibacillusspp. (such as L. sphearicus) insecticidal protein, a Chromobacteriumspp. (such as C. subtsugae or C. piscinae) insecticidal protein, aYersinia spp. (such as Y. entomophaga) insecticidal protein, aPaenibacillus spp. (such as P. propylaea) insecticidal protein, aClostridium spp. (such as C. bifermentans) insecticidal protein, and alignin. In other embodiments, the second agent may be at least oneinsecticidal protein derived from an insecticidal toxin complex (Tc)from Photorhabdus, Xenorhabus, Serratia, or Yersinia. In otherembodiments. The insecticidal protein may be an ADP-ribosyltransferasederived from an insecticidal bacteria, such as Photorhabdus ssp. Instill other embodiments, the insecticidal protein may Axmi205 or derivedfrom Axmi205 (U.S. Pat. Nos. 8,575,425 and 9,394,345, each incorporatedherein by reference). In other embodiments, the insecticidal protein maybe a VIP protein, such as VIP1 and/or VIP2 from B. cereus. In stillother embodiments, the insecticidal protein may be a binary toxinderived from an insecticidal bacteria, such as ISP1A and ISP2A from B.laterosporous or BinA and BinB from L. sphaericus. In still otherembodiments, the insecticidal protein may be engineered or may be ahybrid or chimera of any of the preceding insecticidal proteins.

In some embodiments, the transgenic plant of the invention may compriseat least a second pesticidal agent which is non-proteinaceous. Inpreferred embodiments, the second pesticidal agent is an interfering RNAmolecule. An interfering RNA typically comprises at least a RNA fragmentagainst a target gene, a spacer sequence, and a second RNA fragmentwhich is complementary to the first, so that a double-stranded RNAstructure can be formed. RNA interference (RNAi) occurs when an organismrecognizes double-stranded RNA (dsRNA) molecules and hydrolyzes them.The resulting hydrolysis products are small RNA fragments of about 19-24nucleotides in length, called small interfering RNAs (siRNAs). ThesiRNAs then diffuse or are carried throughout the organism, includingacross cellular membranes, where they hybridize to mRNAs (or other RNAs)and cause hydrolysis of the RNA. Interfering RNAs are recognized by theRNA interference silencing complex (RISC) into which an effector strand(or “guide strand”) of the RNA is loaded. This guide strand acts as atemplate for the recognition and destruction of the duplex sequences.This process is repeated each time the siRNA hybridizes to itscomplementary-RNA target, effectively preventing those mRNAs from beingtranslated, and thus “silencing” the expression of specific genes fromwhich the mRNAs were transcribed. Interfering RNAs are known in the artto be useful for insect control (see, for example, publicationWO2013/192256, incorporated by reference herein). An interfering RNAdesigned for use in insect control produces a non-naturally occurringdouble-stranded RNA, which takes advantage of the native RNAi pathwaysin the insect to trigger down-regulation of target genes that may leadto the cessation of feeding and/or growth and may result in the death ofthe insect pest. The interfering RNA molecule may confer insectresistance against the same target pest as the protein of the invention,or may target a different pest. The targeted insect plant pest may feedby chewing, sucking, or piercing. Interfering RNAs are known in the artto be useful for insect control. In other embodiments, the interferingRNA may confer resistance against a non-insect plant pest, such as anematode pest or a virus pest.

The co-expression of more than one pesticidal agent in the sametransgenic plant can be achieved by making a single recombinant vectorcomprising coding sequences of more than one pesticidal agent in a socalled molecular stack and genetically engineering a plant to containand express all the pesticidal agents in the transgenic plant. Suchmolecular stacks may be also be made by using mini-chromosomes asdescribed, for example in U.S. Pat. No. 7,235,716. Alternatively, atransgenic plant comprising one nucleic acid encoding a first pesticidalagent can be re-transformed with a different nucleic acid encoding asecond pesticidal agent and so forth. Alternatively, a plant, Parent 1,can be genetically engineered for the expression of genes of the presentinvention. A second plant, Parent 2, can be genetically engineered forthe expression of a second pesticidal agent. By crossing Parent 1 withParent 2, progeny plants are obtained which express all the genesintroduced into Parents 1 and 2.

Transgenic plants or seed comprising an insecticidal protein of theinvention can also be treated with an insecticide or insecticidal seedcoating as described in U.S. Pat. Nos. 5,849,320 and 5,876,739, hereinincorporated by reference. Where both the insecticide or insecticidalseed coating and the transgenic plant or seed of the invention areactive against the same target insect, for example a Coleopteran pest ora Diabrotica target pest, the combination is useful (i) in a method forfurther enhancing activity of the composition of the invention againstthe target insect, and (ii) in a method for preventing development ofresistance to the composition of the invention by providing yet anothermechanism of action against the target insect. Thus, the inventionprovides a method of enhancing control of a Diabrotica insect populationcomprising providing a transgenic plant or seed of the invention andapplying to the plant or the seed an insecticide or insecticidal seedcoating to a transgenic plant or seed of the invention.

Even where the insecticidal seed coating is active against a differentinsect, the insecticidal seed coating is useful to expand the range ofinsect control, for example by adding an insecticidal seed coating thathas activity against lepidopteran insects to a transgenic seed of theinvention, which, in some embodiments, has activity against coleopteranand some lepidopteran insects, the coated transgenic seed producedcontrols both lepidopteran and coleopteran insect pests.

Examples of such insecticides and/or insecticidal seed coatings include,without limitation, a carbamate, a pyrethroid, an organophosphate, afriprole, a neonicotinoid, an organochloride, a nereistoxin, or acombination thereof. In another embodiment, the insecticide orinsecticidal seed coating are selected from the group consisting ofcarbofuran, carbaryl, methomyl, bifenthrin, tefluthrin, permethrin,cyfluthrin, lambda-cyhalothrin, cypermethrin, deltamethrin,chlorpyrifos, chlorethoxyfos, dimethoate, ethoprophos, malathion,methyl-parathion, phorate, terbufos, tebupirimiphos, fipronil,acetamiprid, imidacloprid, thiacloprid, thiamethoxam, endosulfan,bensultap, and a combination thereof. Commercial products containingsuch insecticides and insecticidal seed coatings include, withoutlimitation, Furadan® (carbofuran), Lanate® (methomyl, metomil,mesomile), Sevin® (carbaryl), Talstar® (bifenthrin), Force®(tefluthrin), Ammo® (cypermethrin), Cymbush® (cypermethrin), Delta Gold®(deltamethrin), Karate® (lambda-cyhalothrin), Ambush® (permethrin),Pounce® (permethrin), Brigade® (bifenthrin), Capture® (bifenthrin),ProShield® (tefluthrin), Warrior® (lambda-cyhalothrin), Dursban®(chlorphyrifos), Fortress® (chlorethoxyfos), Mocap® (ethoprop), Thimet®(phorate), AAstar® (phorate, flucythinate), Rampart® (phorate), Counter®(terbufos), Cygon® (dimethoate), Dicapthon, Regent® (fipronil), Cruiser®(thiamethoxam), Gaucho® (imidacloprid), Prescribe® (imidacloprid),Poncho® (clothianidin) and Aztec® (cyfluthrin, tebupirimphos).

The present invention also encompasses a composition comprising aneffective insect-controlling amount of an insecticidal protein accordingto the invention. In further embodiments, the composition comprises asuitable agricultural carrier and a polypeptide of the invention withinsecticidal activity. The agricultural carrier may include adjuvants,mixers, enhancers, etc. beneficial for application of an activeingredient, such as a polypeptide of the invention, including apolypeptide comprising an amino acid sequence that is at least 80%, atleast 85%, at least 90%, at least 95%, or 100% identical to of any ofSEQ ID NO: 1-16. Suitable carriers should not be phytotoxic to valuablecrops, particularly at the concentrations employed in applying thecompositions in the presence of crops, and should not react chemicallywith the compounds of the active ingredient herein, namely a polypeptideof the invention, or other composition ingredients. Such mixtures can bedesigned for application directly to crops, or can be concentrates orformulations which are normally diluted with additional carriers andadjuvants before application. They may include inert or activecomponents and can be solids, such as, for example, dusts, powders,granules, water dispersible granules, or wettable powders, or liquids,such as, for example, emulsifiable concentrates, solutions, emulsions orsuspensions. Suitable agricultural carriers may include liquid carriers,for example water, toluene, xylene, petroleum naphtha, crop oil,acetone, methyl ethyl ketone, cyclohexanone, trichloroethylene,perchloroethylene, ethyl acetate, amyl acetate, butyl acetate, propyleneglycol monomethyl ether and diethylene glycol monomethyl ether,methanol, ethanol, isopropanol, amyl alcohol, ethylene glycol, propyleneglycol, glycerine, and the like. Water is generally the carrier ofchoice for the dilution of concentrates. Suitable solid carriers mayinclude talc, pyrophyllite clay, silica, attapulgus clay, kieselguhr,chalk, diatomaceous earth, lime, calcium carbonate, bentonire clay,Fuller's earth, cotton seed hulls, wheat flour, soybean flour, pumice,wood flour, walnut shell flour, lignin, and the like. In anotherembodiment, a polypeptide of the invention may be encapsulated in asynthetic matrix such as a polymer and applied to the surface of a hostsuch as a plant. Ingestion of the host cells by an insect permitsdelivery of the insect control agents to the insect and results in atoxic effect in the insect pest.

In further embodiments, a composition of the invention may be a powder,dust, pellet, granule, spray, emulsion, colloid, or solution. Acomposition of the invention may be prepared by desiccation,lyophilization, homogenization, extraction, filtration, centrifugation,sedimentation, or concentration of a culture of bacterial cells. Acomposition of the invention may comprise at least 1%, about 5%, atleast 10%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 97%, or at least 99% by weight apolypeptide of the invention. A composition of the invention maycomprise at least a second pesticidal agent, which may be insecticidal,nematicidal, fungicidal, or bactericidal. At least a second pesticidalagent may be insecticidal to the same insect as a polypeptide of theinvention or to a different insect. The second pesticidal agent may be apolypeptide. The pesticidal agent may be an interfering RNA. The secondpesticidal agent may be a microorganism, such as a bacteria, whichcomprises a nucleic acid molecule that encodes for a pesticidal agentand/or contains a pesticidal agent such as a polypeptide or interferingRNA. The microorganism may be attenuated, heat-inactivated, orlyophilized. The microorganism may be dead or unable to reproduce. Thesecond pesticidal agent may be an insecticide, for example arbofuran,carbaryl, methomyl, bifenthrin, tefluthrin, permethrin, cyfluthrin,lambda-cyhalothrin, cypermethrin, deltamethrin, chlorpyrifos,chlorethoxyfos, dimethoate, ethoprophos, malathion, methyl-parathion,phorate, terbufos, tebupirimiphos, fipronil, acetamiprid, imidacloprid,thiacloprid, thiamethoxam, endosulfan, bensultap, or a combinationthereof, or a commercial product containing such insecticides andinsecticidal seed coatings as described above.

A composition of the invention, for example a composition comprising apolypeptide of the invention and an agriculturally acceptable carrier,may be used in conventional agricultural methods. An agriculturallyacceptable carrier is a formulation useful for applying a compositioncomprising a polypeptide of the invention to a plant or seed. Forexample, the compositions of the invention may be mixed with waterand/or fertilizers and may be applied preemergence and/or postemergenceto a desired locus by any means, such as airplane spray tanks,irrigation equipment, direct injection spray equipment, knapsack spraytanks, cattle dipping vats, farm equipment used in ground spraying(e.g., boom sprayers, hand sprayers), and the like. The desired locusmay be soil, plants, and the like.

A composition of the invention may be applied to a seed or plantpropagule in any physiological state, at any time between harvest of theseed and sowing of the seed; during or after sowing; and/or aftersprouting. It is preferred that the seed or plant propagule be in asufficiently durable state that it incurs no or minimal damage,including physical damage or biological damage, during the treatmentprocess. A formulation may be applied to the seeds or plant propagulesusing conventional coating techniques and machines, such as fluidizedbed techniques, the roller mill method, rotostatic seed treaters, anddrum coaters.

The present invention also comprises a method for controlling aLepidopteran and/or Coleopteran pest population comprising contactingsaid population with an effective insect-controlling amount of apolypeptide of the invention with insecticidal activity, where thepolypeptide is at least 80%, at least 85%, at least 90%, at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or is 100% identical to any oneof SEQ ID NO: 1-16. Contacting includes members of the pest populationfeeding on or ingesting the polypeptide. The polypeptide may beincorporated into insect diet food or may be expressed in or present onplant tissue which the insect then ingests. In further embodiments,controlling the Lepidopteran and/or Coleopteran pest populationsincludes killing the insects by contacting the insects with an effectiveinsect-controlling amount of a polypeptide of the invention.

The present invention also comprises a method for protecting a plantfrom an insect pest, comprising expressing in a plant or plant cell anucleotide sequence or expression cassette that encodes an insecticidalpolypeptide of the invention. In embodiments, the nucleotide sequence isat least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or is 100% identical to the nucleotide sequenceof SEQ ID NO: 1-16 or encodes a polypeptide comprising an amino acidsequence that is at least 80%, at least 85%, at least 90%, at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or is 100% identical to any ofSEQ ID NO: 1-16. In further embodiments, the plant or plant cellproduces an insecticidal polypeptide having insecticidal activityagainst a Lepidopteran and/or Coleopteran pest.

The present invention also comprises a method for increasing yield in aplant comprising growing in a field a plant, or a seed thereof, havingstably incorporated into its genome a nucleic acid molecule of anexpression cassette of the invention, and wherein said field is infestedwith a pest against which said polypeptide has insecticidal activity.

Once a desired nucleic acid has been transformed into a particular plantspecies, it may be propagated in that species or moved into othervarieties of the same species, particularly including commercialvarieties, using traditional breeding techniques.

In embodiments, a nucleic acid of this invention is expressed intransgenic plants, thus causing the biosynthesis of the correspondinginsecticidal protein in the transgenic plants. In this way, transgenicplants with enhanced resistance to insects, particularly corn rootworm,are generated. For their expression in transgenic plants, the nucleicacids of the invention may optionally be modified and optimized.Although in many cases genes from microbial organisms can be expressedin plants at high levels without modification, low expression intransgenic plants may result from microbial nucleic acids having codonsthat are not preferred in plants. It is known in the art that allorganisms have specific preferences for codon usage, and the codons ofthe nucleic acids described in this invention can be changed to conformwith plant preferences, while maintaining the amino acids encodedthereby. Furthermore, high expression in plants is best achieved fromcoding sequences that have at least about 35% GC content, preferablymore than about 45%, more preferably more than about 50%, and mostpreferably more than about 60%. Microbial nucleic acids that have low GCcontents may express poorly in plants due to the existence of ATTTAmotifs that may destabilize messages, and AATAAA motifs that may causeinappropriate polyadenylation. In embodiments, sequences can be modifiedto account for the specific codon preferences and GC content preferencesof monocotyledons or dicotyledons as these preferences have been shownto differ (Murray et al. Nucl. Acids Res. 17:477-498 (1989)). Inaddition, the nucleic acids are screened for the existence ofillegitimate splice sites that may cause message truncation. All changesrequired to be made within the nucleic acids such as those describedabove can be made using well known techniques of site directedmutagenesis, PCR, and synthetic gene construction, for example, usingthe methods described in the published patent applications EP 0 385 962,EP 0 359 472, and WO 93/07278.

In one embodiment of the invention a coding sequence for an insecticidalprotein of the present invention is made according to the proceduredisclosed in U.S. Pat. No. 5,625,136, herein incorporated by reference.In this procedure, maize preferred codons, i.e., the single codon thatmost frequently encodes that amino acid in maize, are used. The maizepreferred codon for a particular amino acid might be derived, forexample, from known gene sequences from maize. Maize codon usage for 28genes from maize plants is found in Murray et al., Nucleic AcidsResearch 17:477-498 (1989), the disclosure of which is incorporatedherein by reference.

In this manner, the nucleotide sequences can be optimized for expressionin any plant. It is recognized that all or any part of the gene sequencemay be optimized or synthetic. That is, synthetic or partially optimizedsequences may also be used.

For more efficient initiation of translation, sequences adjacent to theinitiating methionine may be modified. For example, they can be modifiedby the inclusion of sequences known to be effective in plants. Joshi hassuggested an appropriate consensus for plants (NAR 15:6643-6653 (1987))and Clontech suggests a further consensus translation initiator(1993/1994 catalog, page 210). These consensus sequences are suitablefor use with the nucleic acids of this invention. In embodiments, thesequences are incorporated into constructions comprising the nucleicacids, up to and including the ATG (whilst leaving the second amino acidunmodified), or alternatively up to and including the GTC subsequent tothe ATG (with the possibility of modifying the second amino acid of thetransgene).

Expression of the nucleic acids in transgenic plants is driven bypromoters that function in plants. The choice of promoter will varydepending on the temporal and spatial requirements for expression, andalso depending on the target species. Thus, expression of the nucleicacids of this invention in leaves, in stalks or stems, in ears, ininflorescences (e.g. spikes, panicles, cobs, etc.), in roots, and/orseedlings is preferred. In many cases, however, protection against morethan one type of insect pest is sought, and thus expression in multipletissues is desirable. Although many promoters from dicotyledons havebeen shown to be operational in monocotyledons and vice versa, ideallydicotyledonous promoters are selected for expression in dicotyledons,and monocotyledonous promoters for expression in monocotyledons.However, there is no restriction to the provenance of selectedpromoters; it is sufficient that they are operational in driving theexpression of the nucleic acids in the desired cell.

In one embodiment promoters are used that are expressed constitutivelyincluding the actin or ubiquitin or cmp promoters or the CaMV 35S and19S promoters. The nucleic acids of this invention can also be expressedunder the regulation of promoters that are chemically regulated.Preferred technology for chemical induction of gene expression isdetailed in the published application EP 0 332 104 (to Ciba-Geigy) andU.S. Pat. No. 5,614,395. A preferred promoter for chemical induction isthe tobacco PR-1a promoter.

In another embodiment a category of promoters which is wound induciblecan be used. Numerous promoters have been described which are expressedat wound sites and also at the sites of phytopathogen infection.Ideally, such a promoter should only be active locally at the sites ofinfection, and in this way the insecticidal proteins of the inventiononly accumulate in cells that need to synthesize the proteins to killthe invading insect pest. Preferred promoters of this kind include thosedescribed by Stanford et al. Mol. Gen. Genet. 215:200-208 (1989), Xu etal. Plant Molec. Biol. 22:573-588 (1993), Logemann et al. Plant Cell1:151-158 (1989), Rohrmeier & Lehle, Plant Molec. Biol. 22:783-792(1993), Firek et al. Plant Molec. Biol. 22:129-142 (1993), and Warner etal. Plant J. 3:191-201 (1993).

Tissue-specific or tissue-preferential promoters useful for theexpression of genes encoding insecticidal proteins of the invention inplants, particularly corn, are those which direct expression in root,pith, leaf or pollen, particularly root. Such promoters, e.g. thoseisolated from PEPC or trpA, are disclosed in U.S. Pat. No. 5,625,136, orMTL, disclosed in U.S. Pat. No. 5,466,785. Both U. S. patents are hereinincorporated by reference in their entirety.

In addition, promoters functional in plastids can be used. Non-limitingexamples of such promoters include the bacteriophage T3 gene 9 5′ UTRand other promoters disclosed in U.S. Pat. No. 7,579,516. Otherpromoters useful with the invention include but are not limited to theS-E9 small subunit RuBP carboxylase promoter and the Kunitz trypsininhibitor gene promoter (Kti3).

In some embodiments of the invention, inducible promoters can be used.Thus, for example, chemical-regulated promoters can be used to modulatethe expression of nucleotide sequences of the invention in a plantthrough the application of an exogenous chemical regulator. Regulationof the expression of nucleotide sequences of the invention via promotersthat are chemically regulated enables the polypeptides of the inventionto be synthesized only when the crop plants are treated with theinducing chemicals. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of a chemical inducesexpression of a nucleotide sequence of the invention, or achemical-repressible promoter, where application of the chemicalrepresses expression of a nucleotide sequence of the invention.

Chemical inducible promoters are known in the art and include, but arenot limited to, the maize In2-2 promoter, which is activated bybenzenesulfonamide herbicide safeners, the maize GST promoter, which isactivated by hydrophobic electrophilic compounds that are used aspre-emergent herbicides, and the tobacco PR-1 a promoter, which isactivated by salicylic acid (e.g., the PR1a system), steroidsteroid-responsive promoters (see, e.g., the glucocorticoid-induciblepromoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88,10421-10425 and McNellis et al. (1998) Plant J. 14, 247-257) andtetracycline-inducible and tetracycline-repressible promoters (see,e.g., Gatz et al. (1991) Mol. Gen. Genet. 227, 229-237, and U.S. Pat.Nos. 5,814,618 and 5,789,156, Lac repressor system promoters,copper-inducible system promoters, salicylate-inducible system promoters(e.g., the PR1a system), glucocorticoid-inducible promoters (Aoyama etal. (1997) Plant J. 11:605-612), and ecdysone-inducible systempromoters.

Other non-limiting examples of inducible promoters include ABA- andturgor-inducible promoters, the auxin-binding protein gene promoter(Schwob et al. (1993) Plant J. 4:423-432), the UDP glucose flavonoidglycosyl-transferase promoter (Ralston et al. (1988) Genetics119:185-197), the MPI proteinase inhibitor promoter (Cordero et al.(1994) Plant J. 6:141-150), and the glyceraldehyde-3-phosphatedehydrogenase promoter (Kohler et al. (1995) Plant Mol. Biol.29:1293-1298; Martinez et al. (1989) J. Mol. Biol. 208:551-565; andQuigley et al. (1989) J. Mol. Evol. 29:412-421). Also included are thebenzene sulphonamide-inducible (U.S. Pat. No. 5,364,780) andalcohol-inducible (Int'l Patent Application Publication Nos. WO 97/06269and WO 97/06268) systems and glutathione S-transferase promoters.Likewise, one can use any of the inducible promoters described in Gatz(1996) Current Opinion Biotechnol. 7:168-172 and Gatz (1997) Annu. Rev.Plant Physiol. Plant Mol. Biol. 48:89-108. Other chemically induciblepromoters useful for directing the expression of the nucleotidesequences of this invention in plants are disclosed in U.S. Pat. No.5,614,395 herein incorporated by reference in its entirety. Chemicalinduction of gene expression is also detailed in the publishedapplication EP 0 332 104 (to Ciba-Geigy) and U.S. Pat. No. 5,614,395. Insome embodiments, a promoter for chemical induction can be the tobaccoPR-1a promoter.

In further aspects, the nucleotide sequences of the invention can beoperably associated with a promoter that is wound inducible or inducibleby pest or pathogen infection (e.g., a insect or nematode plant pest).Numerous promoters have been described which are expressed at woundsites and/or at the sites of pest attack (e.g., insect/nematode feeding)or phytopathogen infection. Ideally, such a promoter should be activeonly locally at or adjacent to the sites of attack, and in this wayexpression of the nucleotide sequences of the invention will be focusedin the cells that are being invaded or fed upon. Such promoters include,but are not limited to, those described by Stanford et al., Mol. Gen.Genet. 215:200-208 (1989), Xu et al. Plant Molec. Biol. 22:573-588(1993), Logemann et al. Plant Cell 1:151-158 (1989), Rohrmeier andLehle, Plant Molec. Biol. 22:783-792 (1993), Firek et al. Plant Molec.Biol. 22:129-142 (1993), Warner et al. Plant J. 3:191-201 (1993), U.S.Pat. Nos. 5,750,386, 5,955,646, 6,262,344, 6,395,963, 6,703,541,7,078,589, 7,196,247, 7,223,901, and U.S. Patent Application Publication2010043102.

In some embodiments of the present invention, a “minimal promoter” or“basal promoter” is used. A minimal promoter is capable of recruitingand binding RNA polymerase II complex and its accessory proteins topermit transcriptional initiation and elongation. In some embodiments, aminimal promoter is constructed to comprise only thenucleotides/nucleotide sequences from a selected promoter that arerequired for binding of the transcription factors and transcription of anucleotide sequence of interest that is operably associated with theminimal promoter including but not limited to TATA box sequences. Inother embodiments, the minimal promoter lacks cis sequences that recruitand bind transcription factors that modulate (e.g., enhance, repress,confer tissue specificity, confer inducibility or repressibility)transcription. A minimal promoter is generally placed upstream (i.e.,5′) of a nucleotide sequence to be expressed. Thus,nucleotides/nucleotide sequences from any promoter useable with thepresent invention can be selected for use as a minimal promoter.

Numerous other sequences can be incorporated into expression cassettesdescribed in this invention. These include sequences that have beenshown to enhance expression such as intron sequences (e.g. from Adhl andbronzel) and viral leader sequences (e.g. from TMV, MCMV and AMV).

It may be preferable to target expression of the nucleic acids of thepresent invention to different cellular localizations in the plant. Insome cases, localization in the cytosol may be desirable, whereas inother cases, localization in some subcellular organelle may bepreferred. Subcellular localization of transgene-encoded enzymes isundertaken using techniques well known in the art. Typically, the DNAencoding the target peptide from a known organelle-targeted gene productis manipulated and fused upstream of the nucleic acid. Many such targetsequences are known for the chloroplast and their functioning inheterologous constructions has been shown. The expression of the nucleicacids of the present invention is also targeted to the endoplasmicreticulum or to the vacuoles of the host cells. Techniques to achievethis are well known in the art.

Vectors suitable for plant transformation are described elsewhere inthis specification. For Agrobacterium-mediated transformation, binaryvectors or vectors carrying at least one T-DNA border sequence aresuitable, whereas for direct gene transfer any vector is suitable andlinear DNA containing only the construction of interest may bepreferred. In the case of direct gene transfer, transformation with asingle DNA species or co-transformation can be used (Schocher et al.Biotechnology 4:1093-1096 (1986)). For both direct gene transfer andAgrobacterium-mediated transfer, transformation is usually (but notnecessarily) undertaken with a selectable marker that may provideresistance to an antibiotic (kanamycin, hygromycin or methotrexate) or aherbicide (basta). Plant transformation vectors comprising the nucleicacid molecules of the present invention may also comprise genes (e.g.phosphomannose isomerase; PMI) which provide for positive selection ofthe transgenic plants as disclosed in U.S. Pat. Nos. 5,767,378 and5,994,629, herein incorporated by reference. The choice of selectablemarker is not, however, critical to the invention.

In embodiments, the nucleic acid can be transformed into the nucleargenome. In another embodiment, a nucleic acid of the present inventionis directly transformed into the plastid genome. A major advantage ofplastid transformation is that plastids are generally capable ofexpressing bacterial genes without substantial codon optimization, andplastids are capable of expressing multiple open reading frames undercontrol of a single promoter. Plastid transformation technology isextensively described in U.S. Pat. Nos. 5,451,513, 5,545,817, and5,545,818, in PCT application no. WO 95/16783, and in McBride et al.(1994) Proc. Nati. Acad. Sci. USA 91, 7301-7305. The basic technique forchloroplast transformation involves introducing regions of clonedplastid DNA flanking a selectable marker together with the gene ofinterest into a suitable target tissue, e.g., using biolistics orprotoplast transformation (e.g., calcium chloride or PEG mediatedtransformation). The 1 to 1.5 kb flanking regions, termed targetingsequences, facilitate homologous recombination with the plastid genomeand thus allow the replacement or modification of specific regions ofthe plastome. Initially, point mutations in the chloroplast 16S rRNA andrps12 genes conferring resistance to spectinomycin and/or streptomycinare utilized as selectable markers for transformation (Svab, Z.,Hajdukiewicz, P., and Maliga, P. (1990) Proc. Nati. Acad. Sci. USA 87,8526-8530; Staub, J. M., and Maliga, P. (1992) Plant Cell 4, 39-45).This resulted in stable homoplasmic transformants at a frequency ofapproximately one per 100 bombardments of target leaves. The presence ofcloning sites between these markers allowed creation of a plastidtargeting vector for introduction of foreign genes (Staub, J. M., andMaliga, P. (1993) EMBO J. 12, 601-606). Substantial increases intransformation frequency are obtained by replacement of the recessiverRNA or r-protein antibiotic resistance genes with a dominant selectablemarker, the bacterial aadA gene encoding the spectinomycin-detoxifyingenzyme aminoglycoside-3′-adenyltransferase (Svab, Z., and Maliga, P.(1993) Proc. Natl. Acad. Sci. USA 90, 913-917). Previously, this markerhad been used successfully for high-frequency transformation of theplastid genome of the green alga Chlamydomonas reinhardtii(Goldschmidt-Clermont, M. (1991) Nucl. Acids Res. 19:4083-4089). Otherselectable markers useful for plastid transformation are known in theart and encompassed within the scope of the invention. Typically,approximately 15-20 cell division cycles following transformation arerequired to reach a homoplastidic state. Plastid expression, in whichgenes are inserted by homologous recombination into all of the severalthousand copies of the circular plastid genome present in each plantcell, takes advantage of the enormous copy number advantage overnuclear-expressed genes to permit expression levels that can readilyexceed 10% of the total soluble plant protein. In a preferredembodiment, a nucleic acid of the present invention is inserted into aplastid-targeting vector and transformed into the plastid genome of adesired plant host. Plants homoplastic for plastid genomes containing anucleic acid of the present invention are obtained, and arepreferentially capable of high expression of the nucleic acid.

EXAMPLES

The invention will be further described by reference to the followingdetailed examples. These examples are provided for the purposes ofillustration only, and are not intended to be limiting unless otherwisespecified. Standard recombinant DNA and molecular cloning techniquesused here are well known in the art and are described by J. Sambrook, etal., Molecular Cloning: A Laboratory Manual, 3d Ed., Cold Spring Harbor,N.Y.: Cold Spring Harbor Laboratory Press (2001); by T. J. Silhavy, M.L. Berman, and L. W. Enquist, Experiments with Gene Fusions, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. (1984) and by Ausubel, F. M.et al., Current Protocols in Molecular Biology, New York, John Wiley andSons Inc., (1988), Reiter, et al., Methods in Arabidopsis Research,World Scientific Press (1992), and Schultz et al., Plant MolecularBiology Manual, Kluwer Academic Publishers (1998).

Example 1: Identification of a Protein with Insecticidal ActivityAgainst Western Corn Rootworm

An insecticidal protein (SEQ ID NO: 1) was identified fromLachnospiraceae bacterium 3_1_57FAA_CT1. An E. coli-optimized version ofthis gene was synthesized (SEQ ID NO: 18) and the gene was cloned into apET29a vector, creating construct pET29a(Lachb). The pET29a(Lachb)construct was transformed into E. coli JM109 (DE3) and proteinexpression was carried out in ZYP-5052 auto-induction media at 25° C.for 24 hours. Lysates were prepared from these cultures and were testedfor bioactivity on Western Corn Rootworm. Briefly, E. coli JM109 (DE3)lysates or purified protein were mixed with an equal volume of heatedartificial insect diet (Bioserv, Inc., Frenchtown, N.J.) in 1.5 mLcentrifuge tubes and then applied to small petri-dishes. After thediet-sample mixture cooled and solidified, 12 WCR larvae were added toeach plate. The plates were sealed and maintained at ambient laboratoryconditions with regard to temperature, lighting and relative humidity.Lysates from E. coli cultures harboring the empty pET29a vector wereused as negative controls. Mortality was assessed on day3 and day 6.

As shown in Table 1, lysate from the culture expressing pET29a(Lachb)showed strong bioactivity against WCR. The Lachnospiraceae bacterium3_1_57FAA_CT1 protein was renamed LachbCRW.

TABLE 1 Insecticidal Activity against Western Corn Rootworm Day 3 Day 6% % Treatment Dead Mortality Dead Mortality Remarks 50 mM Tris pH 8.5, 00 0 0 b 50 mM NaCl JM109/pET29a 1 8 1 8 b JM109/pET29a(Lachb) 7 58 10 83s, m JM109/pET29a(Lachb) 8 67 10 83 s, m Diet alone 0 0 1 8 b s = smalllarvae, m = medium larvae, b = big larvae, vb = very big larvae

Example 2: Variants of LachbCRW Possess Insecticidal Activity AgainstWestern Corn Rootworm

Mutations were introduced into LachbCRW and the protein stability andinsecticidal activity was assayed. LachbCRW residue Y164 was mutated toW (SEQ ID NO: 2) and F (SEQ ID NO: 3). Lysates of these constructs wereanalyzed by Bio-Rad Experion analysis, and it was determined thatLachbCRW Y164W variant produced 3.5-fold more soluble protein ascompared to the wild type protein in E. coli JM109 (DE3) (21% of totalprotein compared to 6% total protein). A second mutation (I169L) wascombined with Y164W (SEQ ID NO: 4) and the protein was produced andpurified and used to determine insecticidal activity.

A bacterial cell lysate was collected from two liters of JM109 (DE3)cells harboring pET29a(LachbCRW Y164W/I169L) that were grown in ZYP-5052auto-induction media. The LachbCRW Y164W/I169L protein was then purifiedand found to be 85-90% pure. The purified protein was tested forefficacy against WCR in a diet-incorporation bioassay, performed asdescribed in Example 1, except mortality was assessed on day 4 and day6. As shown in Table 2, the purified protein demonstrates strongactivity against WCR.

TABLE 2 Insecticidal Activity of LachbCRW Y164W/I169L against WCRLachbCRW Y164W/ Day 4 Day 6 I169L % Re- % Re- (μg/mL) Dead Mortalitymarks Dead Mortality marks 1x PBS only 0 0 b 0 8 b 500 7 58 s, m 12 100s 375 7 58 s, m 12 100 s 250 8 67 s, m 11 92 s 200 9 75 s, m 12 100 s150 9 75 s, m 11 92 s 100 10 83 s, m 12 100 s  50 9 75 s, m 9 75 s, m, bDiet alone 0 0 b 0 0 b s = small larvae, m = medium larvae, b = biglarvae, vb = very big larvae

Several other mutations were combined with Y164W to generate otherdouble mutants of LachbCRW, namely Y164W/Y385W (SEQ ID NO: 5),Y164W/Y400W (SEQ ID NO: 6), Y164W/Y402W (SEQ ID NO: 7), and Y164W/Y431W(SEQ ID NO: 8). Lysates of JM109 (DE3) expressing the LachbCRW doublemutants were made and tested for bioactivity to WCR usingdiet-incorporation assays, performed as described in Example 1 except 10larvae were added to each plate and mortality was assessed at 120 hours(5 days) only. As shown in Table 3, all the double mutants showedbioactivity against WCR.

TABLE 3 Insecticidal Activity of LachbCRW variants against WCR %Treatment Dead Mortality Remarks 50 mM potassium phosphate pH 7.0, 0  0%b 50 mM NaCl JM109/pet29a 4  40% b JM109/LachbCRW Y164W Y385W 10 100% sJM109/LachbCRW Y164W Y400W 10 100% s JM109/LachbCRW Y164W Y402W 10 100%s JM109/LachbCRW Y164W Y431W 10 100% s JM109/LachbCRW Y164W 10 100% s s= small larvae, m = medium larvae, b = big larvae, vb = very big larvae

Other LachbCRW mutants were generated and tested for bioactivity to WCR;these mutants include Y164F (SEQ ID NO: 3), Y164F/I169L (SEQ ID NO: 9),and Y164F/T166S (SEQ ID NO: 10). Lysates of JM109 (DE3) expressing theLachbCRW double mutants were made and tested for bioactivity to WCRusing diet-incorporation assays at a concentration of 0.25 mg Lachbprotein/ml, performed similarly as described in Example 1 exceptmortality was assessed at day 5 and day 7. The WCR bioactivity data forthese mutants is presented in Table 4.

TABLE 4 Insecticidal Activity of LachbCRW variants against WCR Day 5 Day7 % % Treatment mortality Remarks mortality Remarks 50 mM Tris 8.5, 8 b8 b 50 mM NaCl JM109/pET29 empty 0 b 13 b JM109/LachbCRW-wt 33 s, m, b57 s, m, b 0.25 mg/ml JM109/LachbCRW 50 s, m, b 77 s, m, b Y164W 0.25mg/ml JM109/LachbCRW 27 s, m, b 36 s, m, b Y164F 0.25 mg/mlJM109/LachbCRW 62 s, m, b 86 s, m, b Y164F/I169L 0.25 mg/mlJM109/LachbCRW 60 s, m, b 80 s, m, b Y164F/T166S 0.25 mg/ml Diet alone 0b 0 b s = small larvae, m = medium larvae, b = big larvae, vb = very biglarvae

Example 3: LachbCRW Possesses Insecticidal Activity Against NorthernCorn Rootworm

LachbCRW variant Y164W/I169L was purified as in Example 2 and was testedfor efficacy against Northern Corn Rootworm (NCR) in adiet-incorporation assay, performed essentially as described in Example1, except mortality was assessed on day 3 and day 7. LachbCRWY164W/I169L was tested at two different concentrations, 0.6 mg/mL and0.3 mg/mL. The negative control had only 1×PBS. As shown in Table 5,LachbCRW Y164W/I169L variant demonstrates insecticidal activity againstNCR.

TABLE 5 Insecticidal Activity of LachbCRW variant against NCR Day 3 Day7 Treatment Dead Mort % Remarks Dead Mort % Remarks 1x PBS 2 17% mb 325% mb 0.6 mg/mL 8 67% m 8 67% m 0.3 mg/mL 2 17% sm 4 33% sm s = smalllarvae, sm = small/medium larvae, m = medium larvae, mb = medium/biglarvae, b = big larvae, vb = very big larvae

Example 4: LachbCRW Possesses Insecticidal Activity Against SouthernCorn Rootworm

LachbCRW variant Y164W/I169L was purified as in Example 2 and was testedfor efficacy against Southern Corn Rootworm (SCR) in adiet-incorporation assay, performed essentially as described in Example1, except mortality was assessed on days 2, 5, and 7. LachbCRWY164W/I169L was tested at two different concentrations, 0.5 mg/mL and0.25 mg/mL. The negative control had only 1×PBS. As shown in Table 6,LachbCRW Y164W/I169L variant demonstrates some insecticidal activityagainst SCR.

TABLE 6 Insecticidal Activity of LachbCRW variant against SCR Day 2 Day5 Day 8 Mort Mort Mort Treatment Dead % Dead % y Dead % Remarks 1X PBS 00% 0 0% 0  0% b/vb  0.5 mg/ml 0 0% 0 0% 2 17% m/b 0.25 mg/ml 0 0% 0 0% 217% m/b s = small larvae, sm = small/medium larvae, m = medium larvae,mb = medium/big larvae, b = big larvae, vb = very big larvae

Example 5: LachbCRW Possesses Insecticidal Activity Against CryResistant Western Corn Rootworm

To determine if LachbCRW toxicity is through a mode of action separatefrom Cry proteins, LachbCRW variant Y164W/I169L was purified as inExample 2 and was tested for efficacy against a strain of WCR that isresistant to the mCry3A toxin (mCry3A-R) and against a strain of WCRthat is resistant to the eCry3.1Ab toxin (eCry3.1Ab-R).Diet-incorporation assay were performed essentially as described inExample 1, except mortality was assessed on day 4 and day 7. LachbCRWY164W/I169L was tested at two different concentrations, 0.6 mg/mL and0.3 mg/mL. The negative control had only 1×PBS. WCR that is notresistant to mCry3A or eCry3.1Ab (sus) was also assayed. As shown inTable 7, LachbCRW Y164W/I169L variant demonstrates insecticidal activityagainst Cry resistant WCR strains.

TABLE 7 Insecticidal Activity of LachbCRW variant against Cry-R WCR Day4 Day 6 Mort Mort Treatment Dead % Remarks Dead % Remarks sus, 0.6 mg/mL2 17%  10m 5 42% 7m sus, 0.3 mg/mL 3 25%  9m 7 58% 5m sus, 1x PBS 0 0%12mb 1  8% 11mb mCry3A-R, 3 25%  9m 10 83% 2m 0.6 mg/mL mCry3A-R, 0 0%12m 4 33% 8m 0.3 mg/mL mCry3A-R, 0 0% 12mb 3 25% 9mb 1x PBS eCry3.1Ab-R,1 8% 11m 4 33% 8m 0.6 mg/mL eCry3.1Ab-R, 0 0% 12m 2 17% 10mb 0.3 mg/mLeCry3.1Ab-R, 0 0% 12mb 0  0% 12mb 1x PBS s = small larvae, sm =small/medium larvae, m = medium larvae, mb = medium/big larvae, b = biglarvae, vb = very big larvae

Example 6: LachbCRW does not Possess Insecticidal Activity Against FallArmyworm

Lysates of JM109 (DE3)/LachbCRW-wild type were tested for bioactivity tofall armyworm (FAW) in a diet-overlay bioassay, as shown in Table 5.Briefly, E. coli JM109 (DE3) lysates were applied to the surface of anartificial insect diet (Bioserv, Inc., Frenchtown, N.J.) in smallpetri-dishes. After the diet surface dried, twelve FAW larvae were addedto each plate. The plates were sealed and maintained at ambientlaboratory conditions with regard to temperature, lighting and relativehumidity. Lysates from cultures harboring the empty pET29a vector wereused as negative controls. For bioassay experiments utilizing purifiedprotein, 1×PBS was used as the negative control. A positive-controlgroup consisted of larvae exposed to E. coli Bl21* (DE3) lysatesexpressing Vip3D. LachbCRW-wild type was not active to FAW (Table 8).

TABLE 8 Insecticidal Activity of LachbCRW against Fall Armyworm Day 3Day 6 % % Treatment mortality Remarks mortality Remarks 50 mM Tris 8.5,50 mM 0 f 0 f NaCl Jm109/pET29a 0 f 0 f Jm109/LachbCRW 0 f 0 f Vip3D (+)83 nf 92 nf Diet alone 0 f 0 f f = feeding, mf = medium feeding, vsf =very slightly feeding, nf = no feeding, sf = slightly feeding

Example 7: LachbCRW Possesses Insecticidal Activity Against SomeLepidopterans

Lysates of JM109 (DE3)/LachbCRW-wild type were tested for bioactivity ona panel of Lepidopteran insect pests using diet-overlay bioassays.European corn borer (ECB), black cutworm (BCW), corn earworm (CEW),sugar cane borer (SCB), southwestern corn borer (SWCB), soybean looper(sbl), velvet bean caterpillar (VBC), and tobacco budworm (TBW) wereeach tested for LachbCRW Y164W/I169L insecticidal activity by adiet-based assay similar to that of Example 6. 12 larvae were tested foreach experiment, at a LachbCRW protein concentration of 1 μg/cm² . B.thuringiensis strains CO756 (which has multiple Lepidopteran-activetoxins) and AB227 (which is an acrystalliferous strain and contains noLepidopteran-active toxins) were included as positive and negativecontrols, respectively. Insect diet without anything added and with1×PBS added were also included as negative controls. As in Example 6,lysates from bacterial JM109 cultures harboring the empty pET29 vectorwere also used as negative controls.

TABLE 9 Insecticidal Activity of LachbCRW against LepidopteransLachbCRW/ pET29-VC/ Insect Diet C0756 AB227 1xPBS JM109 JM109 ECB 0%100% 0% 0% 0% 0% BCW 0% 100% 0% 0% 17%  8% FAW 0%  83% 8% 0% 0% 8% CEW0% 100% 0% 0% 0% 8% SCB 0% 100% 8% 0% 8% 0% SWCB 0% 100% 17%  8% 17%  0%SBL 0%  0% 0% 8% 0% 0% VBC 0% 100% 0% 0% 0% 17%  TBW 8% 100% 0% 0% 0% 0%

Example 8: Transformation of Maize with LachbCRW Variant

Construct 23075 was generated for LachbCRW maize transformationexperiments. The LachbCRW expression cassette contains the Y164W/I169Lsubstitutions. Construct 23075 comprises an expression cassettecomprising cPMI, which encodes for the selectable marker phosphomannoseisomerase (PMI)) that confers an ability to metabolize mannose (U.S.Pat. Nos. 5,767,378 and 5,994,629), and an expression cassettecomprising a maize codon-optimized nucleotide sequence encoding forLachbCRW Y164W/I169L (SEQ ID NO: 36).

Construct 23075 was transformed into Agrobacterium tumefaciens usingstandard molecular biology techniques known to those skilled in the art.To prepare the Agrobacteria for transformation, cells were cultured inliquid YPC media at 28° C. and 220 rpm overnight. Agrobacteriumtransformation of immature maize embryos was performed essentially asdescribed in Negrotto et al., 2000, (Plant Cell Reports 19: 798-803).For this example, all media constituents are essentially as described inNegrotto et al., supra. However, various media constituents known in theart may be substituted.

Following transformation, selection, and regeneration, plants wereassayed for the presence of the pmi gene and the Lachb Y164W/I169Lcoding sequence (SEQ ID NO: 36) using TaqMan® analysis. Plants were alsotested for the presence of the vector backbone. Plants negative for thevector backbone and comprising one copy of the transgene from construct23075 were transferred to the greenhouse and tested for resistance toWCR.

Example 9: Maize Plants Expressing LachbCRW Variant have InsecticidalActivity Against WCR

Samples of maize root tissue were taken when LachbCRW-expressing maizeevents reached the V3-V4 stage. Maize root tissue was placed in a petridish and then infected with 12 WCR larvae. Root tissue was evaluated forfeeding holes (FH) and scarring damage at day 5. Root tissue fromnon-transformed (null) maize served as the negative control. Expressionof LachbCRW Y164W/I169L in maize events provided protection from WCR ina majority of the LachbCRW transgenic root tissue when compared to thenull sample root tissue (Table 10).

TABLE 10 Insecticidal Activity of Transgenic LachbCRW Y164W/I169L Maizeagainst WCR Plant LachbCRW ELISA (ng/mg) ID Leaf Root Feeding Damage8521 864.54 776.99 4 feeding holes (FH) light scarring 8520 529.541633.03 4 FH, light scarring 8519 293.15 1902.74 3 FH 8517 635.141147.71 6 FH, light scarring 8514 517.47 1298.64 10 FH, medium scarring8511 250.05 693.66 3 FH, light/medium scarring 8509 650.78 335.53 4 FH,light scarring 8506 136.99 634.50 10 FH, light scarring 8503 474.43970.18 5 FH 8499 763.36 1284.51 4 FH 8498 551.89 1229.99 9 FH 8496408.74 1082.70 21 FH, medium/heavy scarring, mite 8494 288.14 833.00 6FH, light scarring 8488 112.18 637.88 at least 10 FH, chewed heavily inplaces, all larvae dead 8486 143.07 633.53 7 FH, medium/heavy scarring8482 348.24 1015.49 11 FH, heavy scarring 8480 389.64 887.25 3 FH 8478330.35 1189.76 3 FH, one end of root chewed 8477 186.19 347.17 10 FH,light scarring 8474 420.58 1740.87 5 FH, light scarring 8473 499.891119.01 5 FH 8469 180.07 663.65 14 FH, heavy scarring 8466 226.13 835.637 FH, one end of root chewed heavily 8461 612.35 534.69 3 FH, lightscarring 8459 290.77 698.30 5 FH, all larvae dead 8457 782.89 703.65 6FH, light scarring 8456 191.58 822.94 7 FH, light scarring 8455 500.231323.00 root completely full of holes, worse than null, all larvae dead8453 359.83 1240.18 4 FH, root chewed to point of breaking in 2-3 spots,all larvae dead 8450 575.22 1251.08 9 FH, light/medium scarring null 15FH, medium/heavy scarring FH = feeding holes

Example 10: LachbCRW Variant in Combination with an Interfering RNA haveInsecticidal Activity Against WCR

LachbCRW and/or a LachbCRW variant, for example LachbCRW variantY164W/I169L, was purified as in Example 2 and dsRNA against an essentialtarget was prepared. The dsRNA and purified protein were tested incombination for efficacy against WCR in a diet-incorporation assay,performed essentially as described in Example 1. Prophetically, thecombination of the dsRNA with the LachbCRW or with the LachbCRW variant,for example LachbCRW variant Y164W/I169L, has insecticidal activityagainst WCR.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof of the description will be suggested topersons skilled in the art and are to be included within the spirit andpurview of this application and the scope of the appended claims.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art thatthis invention pertains. All publications and patent applications areherein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

1. An expression cassette comprising a promoter operably linked to aheterologous nucleic acid molecule comprising: (a) a nucleotide sequenceof any one of SEQ ID NOs: 17-37; (b) a nucleotide sequence that is atleast 80% identical to the nucleotide sequence of any one of SEQ ID NOs:17-37; (c) a nucleotide sequence that encodes a polypeptide, wherein theamino acid sequence of the polypeptide comprises any one of SEQ ID NOs:1-16; (d) a nucleotide sequence that encodes a polypeptide, wherein theamino acid sequence of the polypeptide is at least 80% identical to theamino acid sequence of any one of SEQ ID NOs: 1-16; (e) a nucleotidesequence that is complementary to the nucleotide sequence of any one of(a) to (d) above.
 2. A nucleic acid molecule comprising a nucleotidesequence of any one of SEQ ID NOs: 18-37.
 3. A polypeptide comprising anamino acid sequence of any one of SEQ ID NOs: 2-10 or SEQ ID NOs: 12-16.4. A vector or construct comprising the expression cassette of claim 1.5. A host cell that contains the expression cassette of claim
 1. 6. Thehost cell of claim 5 that is a bacterial cell or a plant cell. 7.(canceled)
 8. A method for producing a polypeptide with insecticidalactivity, comprising culturing the host cell of claim 5 under conditionsin which the nucleic acid molecule encoding the polypeptide isexpressed.
 9. A method of producing a plant or plant part havingenhanced insect resistance as compared to a control plant or plant part,comprising: (a) transforming a nucleic acid molecule comprising theexpression cassette of claim 1 into a plant or plant part; or (b)crossing a first parent plant with a second parent plant, wherein atleast the first parent plant comprises the expression cassette of claim1 and producing at least one progeny plant, wherein the transformedplant or plant part of step (a) or the at least one progeny plant ofstep (b) comprises the expression cassette within its genome andexhibits enhanced insect resistance as compared to a control plant orplant part.
 10. The method of claim 8, wherein the expression cassetteencodes a polypeptide comprising an amino acid sequence that is at least85% identical to SEQ ID NO:
 9. 11.-17. (canceled)
 18. The method ofclaim 9, wherein the enhanced insect resistance is against a coleopteranand/or lepidopteran insect pest.
 19. The method of claim 18, wherein thecoleopteran pest is a Diabrotica species.
 20. The method of claim 19,wherein the the Diabrotica species is selected from the group consistingof Diabrotica virgifera virgifera, Diabrotica barberi and Diabroticaundecimpunctata howardi.
 21. The method of claim 9, wherein the plant orplant part is a monocotyledonous plant.
 22. The method of claim 21,wherein the plant is millet, switchgrass, maize, sorghum, wheat, oat,turf grass, pasture grass, flax, rice, sugarcane, oilseed rape, orbarley.
 23. (canceled)
 24. A transgenic plant comprising the expressioncassette of claim
 1. 25. The transgenic plant of claim 24, wherein saidexpression cassette comprises a nucleotide sequence that has at least(a) 80% identity to any one of SEQ ID NOs: 17 to 37; or (b) 95% identityto SEQ ID NO:36.
 26. (canceled)
 27. The transgenic plant of claim 24wherein said plant is a monocotyledonous plant.
 28. The transgenic plantof claim 27, wherein said monocotyledonous plant is millet, switchgrass,maize, sorghum, wheat, oat, turf grass, pasture grass, flax, rice,sugarcane, oilseed rape, or barley.
 29. (canceled)
 30. The transgenicplant of claim 24, wherein the plant comprises at least one additionaldesired trait, wherein the desired trait is selected from the groupconsisting of insect resistance, abiotic stress tolerance, malesterility, herbicide resistance, bacterial disease resistance, fungaldisease resistance, viral disease resistance, nematode resistance,modified fatty acid metabolism, modified carbohydrate metabolism,production of a commercially valuable enzyme or metabolite, improvednutritional value, improved performance in an industrial process andaltered reproductive capability.
 31. (canceled)
 32. The transgenic plantof claim 30, wherein the additional desired trait is a second pesticidalagent that is an interfering RNA molecule. 33.-38. (canceled)
 39. Amethod for controlling a lepidopteran or coleopteran pest populationcomprising contacting said population with an effectiveinsect-controlling amount of a polypeptide encoded by the expressioncassette of claim
 1. 40.-41. (canceled)